Methods and compositions for modulating car-t activity

ABSTRACT

Disclosed herein are engineered cells and/or hypoimmunogenic cells including engineered and/or hypoimmunogenic stem cells, engineered and/or hypoimmunogenic cells differentiated therefrom, engineered and/or hypoimmunogenic CAR-T cells (primary or differentiated from engineered and/or hypoimmunogenic stem cells) and related methods of their use and generation. Provided herein are engineered and/or hypoimmunogenic cells exhibiting reduced expression of MHC class I and/or MHC class II human leukocyte antigens and T-cell receptors. In some embodiments, such cells also exogenously express one or more tolerogenic factors such as CD47 and one or more chimeric antigen receptors (CAR)s.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Nos. 63/133,171 filed Dec. 31, 2020; 63/136,172filed Jan. 11, 2021; 63/175,003 filed Apr. 14, 2021; 63/255,795 filedOct. 14, 2021; and 63/288,477 filed Dec. 10, 2021, the disclosures ofwhich are herein incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing text copy submitted herewith via EFS-Web wascreated on Mar. 21, 2022, is entitledseq_listing_122864015058US_ST25.TXT, is 93,575 bytes in size and isherein incorporated by reference in its entirety.

SUMMARY

Off-the-shelf CAR-T cells and other therapeutic cells can offeradvantages over autologous cell-based strategies, including ease ofmanufacturing, quality control and avoidance of malignant contaminationand T cell dysfunction. However, the vigorous host-versus-graft immuneresponse against histoincompatible T cells prevents expansion andpersistence of allogeneic CAR-T cells and mitigates the efficacy of thisapproach.

There is substantial evidence in both animal models and human patientsthat hypoimmunogenic cell transplantation is a scientifically feasibleand clinically promising approach to the treatment of numerousdisorders, conditions, and diseases.

There remains a need for novel approaches, compositions and methods forproducing cell-based therapies that avoid detection by the recipient'simmune system.

In some embodiments, provided herein is an engineered cell comprisingreduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M,CIITA, TCR-alpha, and/or TCR-beta relative to a wild-type cell or acontrol cell, the engineered cell further comprising a set of exogenouspolynucleotides comprising a first exogenous polynucleotide encodingCD47 and a second exogenous polynucleotide encoding a chimeric antigenreceptor (CAR), wherein the first and/or second exogenouspolynucleotides are inserted into a specific locus of at least oneallele of the cell. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction,for example, with a vector. In some embodiments, the vector is apseudotyped, self-inactivating lentiviral vector that carries theexogenous polynucleotide. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis virus glycoprotein (VSV-G) envelope, and which carries theexogenous polynucleotide. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using a lentivirus basedviral vector.

In some embodiments, the specific locus is selected from the groupconsisting of a safe harbor or target locus, a B2M locus, a CIITA locus,a TRAC locus and a TRB locus. In many embodiments, the first exogenouspolynucleotide encoding CD47 is inserted into the specific locusselected from the group consisting of a safe harbor or target locus, aB2M locus, a CIITA locus, a TRAC locus and a TRB locus. In someembodiments, the second exogenous polynucleotide encoding the CAR isinserted into the specific locus selected from the group consisting of asafe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locusand a TRB locus. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction,for example, with a vector. In some embodiments, the vector is apseudotyped, self-inactivating lentiviral vector that carries theexogenous polynucleotide. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto different loci. In many embodiments, the first exogenouspolynucleotide encoding CD47 and the second exogenous polynucleotideencoding the CAR are inserted into the same locus. In severalembodiments, the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding the CAR are inserted into theB2M locus. In some embodiments, the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding the CARare inserted into the CIITA locus. In many embodiments, the firstexogenous polynucleotide encoding CD47 and the second exogenouspolynucleotide encoding the CAR are inserted into the TRAC locus. Insome embodiments, the first exogenous polynucleotide encoding CD47 andthe second exogenous polynucleotide encoding the CAR are inserted intothe TRB locus. In some embodiments, the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding the CARare inserted into the safe harbor or target locus. In some embodiments,the safe harbor or target locus is selected from the group consisting ofa CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVS1)gene locus, an albumin gene locus, a SHS231 gene locus, a CLYBL genelocus, a Rosa gene locus (e.g., ROSA26 gene locus), an F3 gene locus(also known as CD142), a MICA gene locus, a MICB gene locus, a LRP1 genelocus (also known as a CD91 gene locus), a HMGB1 gene locus, an ABO genelocus, ad RHD gene locus, a FUT1 locus, and a KDM5D gene locus. Invarious embodiments, the safe harbor or target locus is selected fromthe group consisting of the AAVS1 locus, the CCR5 locus, and the ROSA26locus. In some embodiments, the exogenous polynucleotide is insertedinto at least one allele of the cell using viral transduction, forexample, with a vector. In some embodiments, the vector is apseudotyped, self-inactivating lentiviral vector that carries theexogenous polynucleotide. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the CAR is selected from the group consisting of aCD19-specific CAR and a CD22-specific CAR. In some embodiments, the CARis a bispecific CAR. In some embodiments, the CAR is a CD19-specificCAR. In some embodiments, the CAR is a CD22-specific CAR. In someembodiments, the CAR is a bispecific CAR. In some embodiments, the CARis a CD19/CD22-bispecific CAR.

In many embodiments, the engineered cell does not express HLA-A, HLA-B,and/or HLA-C antigens. In some embodiments, the engineered cell does notexpress B2M. In other embodiments, the engineered cell does not expressHLA-DP, HLA-DQ, and/or HLA-DR antigens. In some embodiments, theengineered cell does not express CIITA. In some embodiments, theengineered cell is selected from the group consisting a pluripotent stemcell, an induced pluripotent stem cell, a T cell differentiated from aninduced pluripotent stem cell, a primary T cell, and a cell derived froma primary T cell, and the engineered cell does not express TCR-alphaand/or TCR-beta.

In many embodiments, the engineered cell is a pluripotent stem cell. Insome embodiments, the engineered cell is an induced pluripotent stemcell.

In some embodiments, the engineered cell is a differentiated cellderived from an induced pluripotent stem cell. In various embodiments,the differentiated cell is selected from the group consisting of an NKcell and a T cell.

In some embodiments, the engineered cell is a cell derived from aprimary T cell. In many embodiments, the cell derived from the primary Tcell is derived from a pool of T cells comprising primary T cells fromone or more donor subjects who are different from a recipient subject.

In some embodiments, the engineered cell is a cell derived from aprimary NK cell. In many embodiments, the cell derived from the primaryNK cell is derived from a pool of NK cells comprising primary NK cellsfrom one or more donor subjects who are different from a recipientsubject.

In some embodiments, the engineered cell retains pluripotency and/orretains differentiation potential.

In many embodiments, following transfer into a first subject, theengineered cell exhibits one or more responses selected from the groupconsisting of (a) a T cell response, (b) an NK cell response, and (c) amacrophage response, that are reduced as compared to a wild-type cellfollowing transfer into a second subject. In some instances, the firstsubject and the second subject are different subjects. In someinstances, the macrophage response is engulfment. In variousembodiments, following transfer into a subject the engineered cellexhibits one or more selected from the group consisting of (a) reducedTH1 activation in the subject, (b) reduced NK cell killing in thesubject, and (c) reduced killing by whole PBMCs in the subject, ascompared to a wild-type cell following transfer into the subject. Inmany embodiments, following transfer into a subject the engineered cellelicits one or more selected from the group consisting of (a) reduceddonor specific antibodies in the subject, (b) reduced IgM or IgGantibodies in the subject, and (c) reduced complement-dependentcytotoxicity (CDC) in a subject, as compared to a wild-type cellfollowing transfer into the subject.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), and/or TRAC^(indel/indel)cell comprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR. In some embodiments, thefirst and/or second exogenous polynucleotides are inserted into at leastone allele of the T cell using viral transduction. In some embodiments,the first and/or second exogenous polynucleotides are inserted into atleast one allele of the T cell using a lentivirus based viral vector. Insome embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), and/or TRAC^(indel/indel)cell comprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the TRAClocus. In many embodiments, the engineered cell is selected from thegroup consisting a pluripotent stem cell, an induced pluripotent stemcell, a T cell differentiated from an induced pluripotent stem cell, aprimary T cell, and a cell derived from a primary T cell, and theengineered cell is a B2M^(indel/indel), CIITA^(indel/indel), and/orTRAC^(indel/indel) cell comprising the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding CARinserted into the TRAC locus. In many embodiments, the engineered cellis selected from the group consisting a pluripotent stem cell, aninduced pluripotent stem cell, a T cell differentiated from an inducedpluripotent stem cell, a primary T cell, and a cell derived from aprimary T cell, and the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), and/or TRAC^(indel/indel) cell comprising firstexogenous polynucleotide encoding CD47 and/or the second exogenouspolynucleotide encoding CAR inserted into the TRB locus. In someembodiments, the engineered cell is selected from the group consisting apluripotent stem cell, an induced pluripotent stem cell, a T celldifferentiated from an induced pluripotent stem cell, a primary T cell,and a cell derived from a primary T cell, and the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), and/or TRAC^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into the TRBlocus. In numerous embodiments, the engineered cell is selected from thegroup consisting a pluripotent stem cell, an induced pluripotent stemcell, a T cell differentiated from an induced pluripotent stem cell, aprimary T cell, and a cell derived from a primary T cell, and theengineered cell is a B2M^(indel/indel), CIITA^(indel/indel), and/orTRAC^(indel/indel) cell comprising first exogenous polynucleotideencoding CD47 and/or the second exogenous polynucleotide encoding CARinserted into the B2M locus. In many embodiments, the engineered cell isselected from the group consisting a pluripotent stem cell, an inducedpluripotent stem cell, a T cell differentiated from an inducedpluripotent stem cell, a primary T cell, and a cell derived from aprimary T cell, and the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), and/or TRAC^(indel/indel) cell comprising the firstexogenous polynucleotide encoding CD47 and the second exogenouspolynucleotide encoding CAR inserted into a B2M locus. In someembodiments, the engineered cell is selected from the group consisting apluripotent stem cell, an induced pluripotent stem cell, a T celldifferentiated from an induced pluripotent stem cell, a primary T cell,and a cell derived from a primary T cell, and the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), and/or TRAC^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the CIITAlocus. In many embodiments, the engineered cell is selected from thegroup consisting a pluripotent stem cell, an induced pluripotent stemcell, a T cell differentiated from an induced pluripotent stem cell, aprimary T cell, and a cell derived from a primary T cell, and theengineered cell is a B2M^(indel/indel), CIITA^(indel/indel), and/orTRAC^(indel/indel) cell comprising the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding CARinserted into a CIITA locus. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), and/or TRB^(indel/indel)cell comprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the TRAClocus. In many embodiments, the engineered cell is selected from thegroup consisting a pluripotent stem cell, an induced pluripotent stemcell, a T cell differentiated from an induced pluripotent stem cell, aprimary T cell, and a cell derived from a primary T cell, and theengineered cell is a B2M^(indel/indel), CIITA^(indel/indel), and/orTRB^(indel/indel) cell comprising the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding CARinserted into the TRAC locus. In many embodiments, the engineered cellis selected from the group consisting a pluripotent stem cell, aninduced pluripotent stem cell, a T cell differentiated from an inducedpluripotent stem cell, a primary T cell, and a cell derived from aprimary T cell, and the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), and/or TRB^(indel/indel) cell comprising firstexogenous polynucleotide encoding CD47 and/or the second exogenouspolynucleotide encoding CAR inserted into the TRB locus. In someembodiments, the engineered cell is selected from the group consisting apluripotent stem cell, an induced pluripotent stem cell, a T celldifferentiated from an induced pluripotent stem cell, a primary T cell,and a cell derived from a primary T cell, and the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), and/or TRB^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into the TRBlocus. In numerous embodiments, the engineered cell is selected from thegroup consisting a pluripotent stem cell, an induced pluripotent stemcell, a T cell differentiated from an induced pluripotent stem cell, aprimary T cell, and a cell derived from a primary T cell, and theengineered cell is a B2M^(indel/indel), CIITA^(indel/indel), and/orTRB^(indel/indel) cell comprising first exogenous polynucleotideencoding CD47 and/or the second exogenous polynucleotide encoding CARinserted into the B2M locus. In many embodiments, the engineered cell isselected from the group consisting a pluripotent stem cell, an inducedpluripotent stem cell, a T cell differentiated from an inducedpluripotent stem cell, a primary T cell, and a cell derived from aprimary T cell, and the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), and/or TRB^(indel/indel) cell comprising the firstexogenous polynucleotide encoding CD47 and the second exogenouspolynucleotide encoding CAR inserted into a B2M locus. In someembodiments, the engineered cell is selected from the group consisting apluripotent stem cell, an induced pluripotent stem cell, a T celldifferentiated from an induced pluripotent stem cell, a primary T cell,and a cell derived from a primary T cell, and the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), and/or TRB^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the CIITAlocus. In many embodiments, the engineered cell is selected from thegroup consisting a pluripotent stem cell, an induced pluripotent stemcell, a T cell differentiated from an induced pluripotent stem cell, aprimary T cell, and a cell derived from a primary T cell, and theengineered cell is a B2M^(indel/indel), CIITA^(indel/indel), and/orTRB^(indel/indel) cell comprising the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding CARinserted into a CIITA locus. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), and/or TRB^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the TRAClocus. In many embodiments, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), and/or TRB^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into the TRAClocus. In many embodiments, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), and/or TRB^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the TRBlocus. In some embodiments, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), and/or TRB^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into the TRBlocus. In numerous embodiments, the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel), and/orTRB^(indel/indel) cell comprising first exogenous polynucleotideencoding CD47 and/or the second exogenous polynucleotide encoding CARinserted into the B2M locus. In many embodiments, the engineered cell isa B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel), and/orTRB^(indel/indel) cell comprising the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding CARinserted into a B2M locus. In some embodiments, the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel), and/orTRB^(indel/indel) cell comprising first exogenous polynucleotideencoding CD47 and/or the second exogenous polynucleotide encoding CARinserted into the CIITA locus. In many embodiments, the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel), and/orTRB^(indel/indel) cell comprising the first exogenous polynucleotideencoding CD47 and the second exogenous polynucleotide encoding CARinserted into a CIITA locus. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, provided is an engineered cell comprising reducedexpression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA,TCR-alpha, and/or TCR-beta relative to a wild-type cell or a controlcell.

In some embodiments, the engineered cell does not express HLA-A, HLA-Band/or HLA-C antigens. In many embodiments, the engineered cell does notexpress CIITA.

In many embodiments, the engineered cell does not express HLA-DP,HLA-DQ, and/or HLA-DR antigens. In some embodiments, the engineered celldoes not express B2M.

In many embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered celldoes not express TCR-alpha. In many embodiments, the engineered cell isselected from the group consisting a pluripotent stem cell, an inducedpluripotent stem cell, a T cell differentiated from an inducedpluripotent stem cell, a primary T cell, and a cell derived from aprimary T cell, and the engineered cell does not express TCR-beta.

In various embodiments, the engineered cell overexpresses CD47 relativeto a wild-type cell or a control cell.

In some embodiments, the engineered cell is a pluripotent stem cell. Inmany embodiments, the engineered cell is an induced pluripotent stemcell.

In many embodiments, the engineered cell is a differentiated cellderived from an induced pluripotent stem cell. In some embodiments, thedifferentiated cell is selected from the group consisting of an NK celland a T cell.

In many embodiments, the engineered cell is a cell derived from aprimary T cell. In several embodiments, the cell derived from theprimary T cell is derived from a pool of T cells comprising primary Tcells from one or more donor subjects who are different from a recipientsubject.

In various embodiments, the engineered cell retains pluripotency and/orretains differentiation potential.

In some embodiments, following transfer into a subject the engineeredcell elicits one or more response selected from the group consisting of(a) a T cell response, (b) an NK cell response, and (c) a macrophageresponse, that are reduced as compared to a wild-type cell followingtransfer into a second subject. In some instances, the first subject andthe second subject are different subjects. In some instances, themacrophage response is engulfment.

In various embodiments, following transfer into a subject the engineeredcell exhibits one or more selected from the group consisting of (a)reduced TH1 activation in the subject, (b) reduced NK cell killing inthe subject, and (c) reduced killing by whole PBMCs in the subject, ascompared to a wild-type cell following transfer into the subject. Inmany embodiments, following transfer into a subject the engineered cellelicits one or more selected from the group consisting of (a) reduceddonor specific antibodies in the subject, (b) reduced IgM or IgGantibodies in the subject, and (c) reduced complement-dependentcytotoxicity (CDC) in a subject, as compared to a wild-type cellfollowing transfer into the subject.

In some embodiments, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), and/or TRB^(indel/indel) cell.In some instances, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), and/or TRB^(indel/indel)primary T cell. In some instances, the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel), and/orTRB^(indel/indel) T cell differentiated from a hypoimmunogenic inducedpluripotent stem cell. In some embodiments, the engineered cell isselected from the group consisting a pluripotent stem cell, an inducedpluripotent stem cell, a T cell differentiated from an inducedpluripotent stem cell, a primary T cell, and a cell derived from aprimary T cell, and the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell. In some instances, theengineered cell is selected from the group consisting a pluripotent stemcell, an induced pluripotent stem cell, a T cell differentiated from aninduced pluripotent stem cell, a primary T cell, and a cell derived froma primary T cell, and the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) primary T cell. In someembodiments, the engineered cell is selected from the group consisting apluripotent stem cell, an induced pluripotent stem cell, a T celldifferentiated from an induced pluripotent stem cell, a primary T cell,and a cell derived from a primary T cell, and the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), TRB^(indel/indel) cell. In someinstances, the engineered cell is selected from the group consisting apluripotent stem cell, an induced pluripotent stem cell, a T celldifferentiated from an induced pluripotent stem cell, a primary T cell,and a cell derived from a primary T cell, and the engineered cell is aB2M^(indel/indel), CIITA^(indel/indel), TRB^(indel/indel) primary Tcell. In some instances, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), and/or TRAC^(indel/indel) T cell differentiatedfrom a hypoimmunogenic induced pluripotent stem cell. In someembodiments, the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), and/or TRB^(indel/indel) cell. In some instances,the engineered cell is a B2M^(indel/indel), CIITA^(indel/indel), and/orTRB^(indel/indel) primary T cell. In some instances, the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), and/or TRB^(indel/indel) Tcell differentiated from a hypoimmunogenic induced pluripotent stemcell.

In some embodiments, the engineered cell is a hypoimmunogenic cell.

In some embodiments, provided is a pharmaceutical composition comprisinga population of any of the engineered cells described herein and apharmaceutically acceptable additive, carrier, diluent or excipient.

In some embodiments, the pharmaceutically acceptable additive, carrier,diluent or excipient comprises one or more selected from the groupconsisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, humanserum albumin (HSA), dimethylsulfoxide (DMSO), and a combinationthereof. In some embodiments, the pharmaceutical composition furthercomprises a pharmaceutically acceptable buffer. In some embodiments, thepharmaceutically acceptable buffer is neutral buffer saline or phosphatebuffered saline.

In some embodiments, provided is a pharmaceutical composition comprisinga population of any of the engineered cells described herein, a basesolution of CryoStor® CSB at a concentration of about 70-80% w/w, andone or more of about 20-30% w/w PlasmaLyte-A™ about 0.3-5.3% w/v humanserum albumin (HSA), about 0-20% v/v dimethylsulfoxide (DMSO), and about100-400 mM trehalose.

In some embodiments, provided is a pharmaceutical composition comprisinga population of any of the engineered cells described herein, a basesolution of PlasmaLyte-A™ at a concentration of about 20-30% w/w, andone or more of about 70-80% w/w CryoStor® CSB, about 0.3-5.3% w/v humanserum albumin (HSA), about 0-20% v/v dimethylsulfoxide (DMSO), and about100-400 mM trehalose.

In some embodiments, provided is a pharmaceutical composition comprisinga population of any of the engineered cells described herein, about0.3-5.3% w/v human serum albumin (HSA), and one or more of about 70-80%w/w CryoStor® CSB, about 20-30% w/w PlasmaLyte-A™, about 0-20% v/vdimethylsulfoxide (DMSO), and about 100-400 mM trehalose.

In some embodiments, provided is a pharmaceutical composition comprisinga population of any of the engineered cells described herein, about0-20% v/v dimethylsulfoxide (DMSO), and one or more of about 70-80% w/wCryoStor® CSB, about 20-30% w/w PlasmaLyte-A™, about 0.3-5.3% w/v humanserum albumin (HSA), and about 100-400 mM trehalose.

In some embodiments, provided is a pharmaceutical composition comprisinga population of any of the engineered cells described herein, about100-400 mM trehalose, and one or more of about 70-80% w/w CryoStor® CSB,about 20-30% w/w PlasmaLyte-A™, about 0.3-5.3% w/v human serum albumin(HSA), and about 0-20% v/v dimethylsulfoxide (DMSO).

In some embodiments, the pharmaceutical composition comprises about 75%w/w of CryoStor® CSB. In some embodiments, the pharmaceuticalcomposition comprises about 25% w/w of PlasmaLyte-A™. In someembodiments, the pharmaceutical composition comprises about 0.3% w/v ofHSA. In some embodiments, the pharmaceutical composition comprises about7.5% v/v of DMSO.

In some embodiments, provided is a pharmaceutical composition comprisinga population of any of the engineered cells described herein, a basesolution of CryoStor® CSB at a concentration of about 75% w/w, about 25%w/w PlasmaLyte-A™, about 0.3% w/v human serum albumin (HSA), and about7.5% v/v dimethylsulfoxide (DMSO).

In some embodiments, the population of the engineered cells is up toabout 8.0×10⁸ cells. In many embodiments, the population of theengineered cells is up to about 6.0×10⁸ cells. In other embodiments, thepopulation of the engineered cells is from about 1.0×10⁶ to about2.5×10⁸ cells. In some embodiments, the population of the engineeredcells is from about 2.0×10⁶ to about 2.0×10⁸ cells.

In various embodiments, the population of the engineered cells rangesfrom about 5 ml to about 80 ml. In many embodiments, the population ofthe engineered cells ranges from about 10 ml to about 70 ml. In someembodiments, the population of the engineered cells ranges from about 10ml to about 50 ml.

In some embodiments, the composition is formulated for administration ina single dose. In many embodiments, the composition is formulated foradministration in up to three doses.

In some embodiments, the composition is formulated for administration ofa single dose to a subject takes a duration of time of about 60 minutesor less. In many embodiments, the composition is formulated foradministration of a single dose to a subject takes a duration of time ofabout 30 minutes or less.

In some embodiments, the population of engineered cells of thepharmaceutical composition or progeny thereof exhibit at least 40%survival in a subject after 10 days following administration. In variousembodiments, the population of engineered cells of the pharmaceuticalcomposition or progeny thereof exhibit at least 80% survival in asubject after about 2 weeks following administration. In severalembodiments, the population of engineered cells of the pharmaceuticalcomposition or progeny thereof exhibit at least 100% survival in asubject after about 3 weeks following administration. In manyembodiments, the population of engineered cells of the pharmaceuticalcomposition or progeny thereof exhibit at least 150% survival in asubject after about 4 weeks following administration.

In another embodiment, provided is a dosage regimen for treating adisease or disorder in a subject comprising administration of apharmaceutical composition comprising a population of any of theengineered cells described herein and a pharmaceutically acceptableadditive, carrier, diluent or excipient, wherein the pharmaceuticalcomposition is administered in about 1-3 doses.

In some embodiments, the pharmaceutical composition administered is upto about 6.0×10⁸ cells in about 1-3 doses. In some embodiments, thepharmaceutical composition administered is from about 0.6×10⁶ to about6.0×10⁸ cells in about 1-3 doses. In some embodiments, thepharmaceutical composition administered is from about 0.2×10⁶ to about5.0×10⁶ cells per kg of the subject's body weight in about 1-3 doses, ifthe subject has a body weight of 50 kg or less. In some embodiments, thepharmaceutical composition administered is from about 0.1×10⁸ to about2.5×10⁸ cells in about 1-3 doses, if the subject has a body weightgreater than 50 kg. In some embodiments, the pharmaceutical compositionadministered is from about 2.0×10⁶ cells per kg of the subject's bodyweight and up to about 2.×10⁸ cells in about 1-3 doses.

In some embodiments, the administration of a single dose to the subjecttakes a duration of time of about 60 minutes or less. In someembodiments, the administration of a single dose to the subject takes aduration of time of about 30 minutes or less.

In some embodiments, the pharmaceutically acceptable additive, carrier,diluent or excipient comprises one or more selected from the groupconsisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, humanserum albumin (HSA), dimethylsulfoxide (DMSO), and a combinationthereof.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable buffer. In some embodiments, thepharmaceutically acceptable buffer is neutral buffer saline or phosphatebuffered saline.

In some embodiments, after the administration of the pharmaceuticalcomposition, the population of cells or progeny thereof are present inthe subject up to 9 months. In some embodiments, after theadministration of the pharmaceutical composition, the population ofcells or progeny thereof are present in the subject at least 2 years ormore.

In some embodiments, after the administration of the pharmaceuticalcomposition, the population of engineered cells or progeny thereofexhibit at least 40% survival in a subject after about 10 days followingadministration. In some embodiments, after the administration of thepharmaceutical composition, the population of engineered cells orprogeny thereof exhibit at least 80% survival in a subject after about 2weeks following administration. In some embodiments, after theadministration of the pharmaceutical composition, the population ofengineered cells or progeny thereof exhibit at least 100% survival in asubject after about 3 weeks following administration. In someembodiments, after the administration of the pharmaceutical composition,the population of engineered cells or progeny thereof exhibit at least150% survival in a subject after about 4 weeks following administration.

In some embodiments, the administration of 2-3 doses to the subjectoccurs such that each dose is administered ranging from 1 to 24 hoursapart. In some embodiments, the administration of 2-3 doses to thesubject occurs such that each dose is administered ranging from 1 to 28days apart. In some embodiments, the administration of 2-3 doses to thesubject occurs such that each dose is administered ranging from 1 to 6weeks apart. In some embodiments, the administration of 2-3 doses to thesubject occurs such that each dose is administered ranging from 1 to 12months or more apart.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising (i) an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta, the engineered cell further comprising a set ofexogenous polynucleotides encoding CD47 and a chimeric antigen receptor(CAR). In some embodiments, the set of exogenous polynucleotides areinserted into at least one allele of the T cell using viraltransduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the set of exogenous polynucleotides. In some embodiments, thevector is a self-inactivating lentiviral vector pseudotyped with avesicular stomatitis VSV-G envelope, and which carries the set ofexogenous polynucleotides. In some embodiments, set of exogenouspolynucleotides are inserted into at least one allele of the T cellusing a lentivirus based viral vector. In some embodiments, the set ofexogenous polynucleotides are inserted into a safe harbor or targetlocus of at least one allele of the cell; and (ii) a pharmaceuticallyacceptable additive, carrier, diluent or excipient, wherein thepharmaceutical composition comprises up to about 6.0×10⁸ cells. In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction, for example, with a vector.In some embodiments, the vector is a pseudotyped, self-inactivatinglentiviral vector that carries the exogenous polynucleotide. In someembodiments, the vector is a self-inactivating lentiviral vectorpseudotyped with a vesicular stomatitis VSV-G envelope, and whichcarries the exogenous polynucleotide. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using a lentivirus basedviral vector.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising (i) an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta, the engineered cell further comprising a set ofexogenous polynucleotides encoding CD47 and a chimeric antigen receptor(CAR). In some embodiments, the set of exogenous polynucleotides areinserted into at least one allele of the T cell using viraltransduction. In some embodiments, set of exogenous polynucleotides areinserted into at least one allele of the T cell using a lentivirus basedviral vector. In some embodiments, the set of exogenous polynucleotidesare inserted into a safe harbor or target locus of at least one alleleof the cell; and (ii) a pharmaceutically acceptable additive, carrier,diluent or excipient, wherein the pharmaceutical composition isadministered in 1-3 doses. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising (i) an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta, the engineered cell further comprising a set ofexogenous polynucleotides encoding CD47 and a chimeric antigen receptor(CAR), wherein the set of exogenous polynucleotides are inserted into asafe harbor or target locus of at least one allele of the cell; and (ii)a pharmaceutically acceptable additive, carrier, diluent or excipient,wherein a dose of the pharmaceutical composition is administered for aduration of time of about 60 minutes or less. In some embodiments, theexogenous polynucleotide is inserted into at least one allele of thecell using viral transduction, for example, with a vector. In someembodiments, the vector is a pseudotyped, self-inactivating lentiviralvector that carries the exogenous polynucleotide. In some embodiments,the vector is a self-inactivating lentiviral vector pseudotyped with avesicular stomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising (i) an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta; and (ii) a pharmaceutically acceptable additive,carrier, diluent or excipient, wherein the pharmaceutical compositioncomprises up to about 6.0×10⁸ cells.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising (i) an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta; and (ii) a pharmaceutically acceptable additive,carrier, diluent or excipient, wherein the pharmaceutical composition isadministered in 1-3 doses.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising (i) an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta; and (ii) a pharmaceutically acceptable additive,carrier, diluent or excipient, wherein a dose of the pharmaceuticalcomposition is administered for a duration of 60 minutes or less.

In some embodiments, provided is a method of treating a cancer in asubject comprising administration of any of the engineered cellsdescribed herein or any of the pharmaceutical compositions describedherein or any of the dosage regimens described herein to the subject. Insome embodiments, the cancer is a CD19⁺ cancer.

In some embodiments, provided is a method of preventing T cellexhaustion or treating a disease in a subject comprising administrationof any of the engineered cells described herein to the subject, whereinthe CAR is a CD19/CD22-bispecific CAR.

In some embodiments, provided herein is a method of preventing T cellexhaustion or treating a disease in a subject comprising: (i)administration of a first dosage regimen comprising a first populationof any of the engineered cells described herein to the subject at afirst timepoint, and (ii) administration of a second dosage regimencomprising a second population of any of the engineered cells describedherein to the subject at a second timepoint, wherein the first dosageregimen and the second dosage regimen are different.

In some embodiments, provided herein is a method of preventing T cellexhaustion or treating a disease in a subject comprising: (i)administration of a first dosage regimen comprising a first populationof any of the engineered cells described herein to the subject at afirst timepoint, and (ii) administration of a second dosage regimencomprising a second population of any of the engineered cells describedherein to the subject at a second timepoint, wherein the firstpopulation of engineered cells and the second population of engineeredcells both comprise the same chimeric antigen receptor.

In some embodiments, provided herein is a method of preventing T cellexhaustion or treating a disease in a subject comprising: (i)administration of a first dosage regimen comprising a first populationof any of the engineered cells described herein to the subject at afirst timepoint, and (ii) administration of a second dosage regimencomprising a second population of any of the engineered cells describedherein to the subject at a second timepoint, wherein the firstpopulation of engineered cells and the second population of engineeredcells both comprise different chimeric antigen receptors.

In some embodiments, provided herein is a method of preventing T cellexhaustion or treating a disease in a subject comprising: (i)administration of a first dosage regimen comprising a first populationof any of the engineered cells described herein to the subject at afirst timepoint, and (ii) administration of a second dosage regimencomprising a second population of any of the engineered cells describedherein to the subject at a second timepoint, wherein the engineeredcells of the first population comprise a first chimeric antigen receptorthat binds a first antigen and the engineered cells of the secondpopulation comprise a second chimeric antigen receptor that binds asecond antigen, and wherein the first antigen and the second antigen arethe same.

In some embodiments, provided herein is a method of preventing T cellexhaustion or treating a disease in a subject comprising: (i)administration of a first dosage regimen comprising a first populationof any of the engineered cells described herein to the subject at afirst timepoint, and (ii) administration of a second dosage regimencomprising a second population of any of the engineered cells describedherein to the subject at a second timepoint, wherein the engineeredcells of the first population comprise a first chimeric antigen receptorthat binds a first antigen and the engineered cells of the secondpopulation comprise a second chimeric antigen receptor that binds asecond antigen, and wherein the first antigen and the second antigen aredifferent.

Provided herein are non-activated T cells comprising reduced expressionof HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type T cell, and a first exogenouspolynucleotide encoding a chimeric antigen receptor (CAR). In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction, for example, with a vector.In some embodiments, the vector is a pseudotyped, self-inactivatinglentiviral vector that carries the exogenous polynucleotide. In someembodiments, the vector is a self-inactivating lentiviral vectorpseudotyped with a vesicular stomatitis VSV-G envelope, and whichcarries the exogenous polynucleotide. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using a lentivirus basedviral vector.

In some embodiments, the non-activated T cell is a primary T cell. Inother embodiments, the non-activated T cell is differentiated from theengineered cells of the present technology.

In some embodiments, the T cell is a CD8⁺ T cell.

In some embodiments, the non-activated T cell has not been treated withan anti-CD3 antibody, an anti-CD28 antibody, a T cell activatingcytokine, or a soluble T cell costimulatory molecule.

In some embodiments, the anti-CD3 antibody is OKT3. In some embodiments,the anti-CD28 antibody is CD28.2. In some embodiments, the T cellactivating cytokine is selected from the group of T cell activatingcytokines consisting of IL-2, IL-7, IL-15, and IL-21. In someembodiments, the soluble T cell costimulatory molecule is selected fromthe group of soluble T cell costimulatory molecules consisting of ananti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, ananti-CD137L antibody, and an anti-ICOS-L antibody.

In some embodiments, the non-activated T cell does not expressactivation markers.

In some embodiments, the non-activated T cell expresses CD3 and CD28,and wherein the CD3 and/or CD28 are inactive.

In some embodiments, the first exogenous polynucleotide is carried by alentiviral vector comprising a CD8 binding agent.

In some embodiments, the non-activated T cell further comprises a secondexogenous polynucleotide encoding CD47.

In some embodiments, the first and/or second exogenous polynucleotidesare inserted into a specific locus of at least one allele of the T cell.In some embodiments, the first and/or second exogenous polynucleotidesare inserted into at least one allele of the T cell using viraltransduction. In some embodiments, the first and/or second exogenouspolynucleotides are inserted into at least one allele of the T cellusing a lentivirus based viral vector. In some embodiments, the vectoris a pseudotyped, self-inactivating lentiviral vector that carries thefirst and/or second exogenous polynucleotides. In some embodiments, thevector is a self-inactivating lentiviral vector pseudotyped with avesicular stomatitis VSV-G envelope, and which carries the first and/orsecond exogenous polynucleotides. In some embodiments, the specificlocus is selected from the group consisting of a safe harbor or targetlocus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. Insome embodiments, the second exogenous polynucleotide encoding CD47 isinserted into the specific locus selected from the group consisting of asafe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locusand a TRB locus. In some embodiments, the first exogenous polynucleotideencoding the CAR is inserted into the specific locus selected from thegroup consisting of a safe harbor or target locus, a B2M locus, a CIITAlocus, a TRAC locus and a TRB locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into different loci. Insome embodiments, the second exogenous polynucleotide encoding CD47 andthe first exogenous polynucleotide encoding the CAR are inserted intothe same locus. In some embodiments, the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding the CARare inserted into the B2M locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into the CIITA locus. Insome embodiments, the second exogenous polynucleotide encoding CD47 andthe first exogenous polynucleotide encoding the CAR are inserted intothe TRAC locus. In some embodiments, the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding the CARare inserted into the TRB locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into the safe harbor ortarget locus. In some embodiments, the safe harbor or target locus isselected from the group consisting of a CCR5 gene locus, a CXCR4 genelocus, a PPP1R12C (also known as AAVS1) gene locus, an albumin genelocus, a SHS231 gene locus, a CLYBL gene locus, a Rosa gene locus (e.g.,ROSA26 gene locus), an F3 gene locus (also known as CD142), a MICA genelocus, a MICB gene locus, a LRP1 gene locus (also known as a CD91 genelocus), a HMGB1 gene locus, an ABO gene locus, ad RHD gene locus, a FUT1locus, and a KDM5D gene locus. In some embodiments, the safe harbor ortarget locus is selected from the group consisting of the AAVS1 locus,the CCR5 locus, and the ROSA26 locus. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the CAR is selected from the group consisting of aCD19-specific CAR and a CD22-specific CAR. In some embodiments, the CARis a bispecific CAR. In some embodiments, the bispecific CAR is aCD19/CD22-bispecific CAR.

In some embodiments, the non-activated T cell does not express HLA-A,HLA-B, and/or HLA-C antigens. In some embodiments, the non-activated Tcell does not express B2M. In some embodiments, the non-activated T celldoes not express HLA-DP, HLA-DQ, and/or HLA-DR antigens. In someembodiments, the non-activated T cell does not express CIITA. In someembodiments, the non-activated T cell does not express TCR-alpha andTCR-beta.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the TRAC locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into the TRAC locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the TRB locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into the TRB locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the B2M locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into a B2M locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the CIITA locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into a CIITA locus.

In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using viral transduction, for example, witha vector. In some embodiments, the vector is a pseudotyped,self-inactivating lentiviral vector that carries the exogenouspolynucleotide. In some embodiments, the vector is a self-inactivatinglentiviral vector pseudotyped with a vesicular stomatitis VSV-Genvelope, and which carries the exogenous polynucleotide. In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction. In some embodiments, theexogenous polynucleotide is inserted into at least one allele of thecell using a lentivirus based viral vector.

Provided herein are engineered T cells comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type T cell, wherein the engineered Tcell further comprises a first exogenous polynucleotide encoding achimeric antigen receptor (CAR) carried by a lentiviral vector. Providedherein are engineered T cells comprising reduced expression of HLA-A,HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and/orTCR-beta relative to a wild-type T cell, wherein the engineered T cellfurther comprises a first exogenous polynucleotide encoding a chimericantigen receptor (CAR) carried by a lentiviral vector that comprises aCD8 binding agent. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction,for example, with a vector. In some embodiments, the vector is apseudotyped, self-inactivating lentiviral vector that carries theexogenous polynucleotide. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the engineered T cell is a primary T cell. In otherembodiments, the engineered T cell is differentiated from the engineeredcell of the present technology. In some embodiments, the T cell is aCD8⁺ T cell.

In some embodiments, the engineered T cell has not been treated with ananti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine,or a soluble T cell costimulatory molecule. In some embodiments, theanti-CD3 antibody is OKT3, wherein the anti-CD28 antibody is CD28.2,wherein the T cell activating cytokine is selected from the group of Tcell activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21,and wherein soluble T cell costimulatory molecule is selected from thegroup of soluble T cell costimulatory molecules consisting of ananti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, ananti-CD137L antibody, and an anti-ICOS-L antibody.

In some embodiments, the engineered T cell does not express activationmarkers. In some embodiments, the engineered T cell expresses CD3 andCD28, and wherein the CD3 and/or CD28 are inactive.

In some embodiments, the engineered T cell further comprises a secondexogenous polynucleotide encoding CD47. In some embodiments, the firstand/or second exogenous polynucleotides are inserted into a specificlocus of at least one allele of the T cell. In some embodiments, thespecific locus is selected from the group consisting of a safe harbor ortarget locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.In some embodiments, the second exogenous polynucleotide encoding CD47is inserted into the specific locus selected from the group consistingof a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAClocus and a TRB locus. In some embodiments, the first exogenouspolynucleotide encoding the CAR is inserted into the specific locusselected from the group consisting of a safe harbor or target locus, aB2M locus, a CIITA locus, a TRAC locus and a TRB locus. In someembodiments, the second exogenous polynucleotide encoding CD47 and thefirst exogenous polynucleotide encoding the CAR are inserted intodifferent loci. In some embodiments, the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding the CARare inserted into the same locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into the B2M locus, theCIITA locus, the TRAC locus, the TRB locus, or the safe harbor or targetlocus. In some embodiments, the safe harbor or target locus is selectedfrom the group consisting of a CCR5 gene locus, a CXCR4 gene locus, aPPP1R12C (also known as AAVS1) gene locus, an albumin gene locus, aSHS231 gene locus, a CLYBL gene locus, a Rosa gene locus (e.g., ROSA26gene locus), an F3 gene locus (also known as CD142), a MICA gene locus,a MICB gene locus, a LRP1 gene locus (also known as a CD91 gene locus),a HMGB1 gene locus, an ABO gene locus, ad RHD gene locus, a FUT1 locus,and a KDM5D gene locus. In some embodiments, the safe harbor or targetlocus is selected from the group consisting of the AAVS1 locus, the CCR5locus, and the ROSA26 locus. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the CAR is selected from the group consisting of aCD19-specific CAR and a CD22-specific CAR.

In some embodiments, the engineered T cell does not express HLA-A,HLA-B, and/or HLA-C antigens, wherein the engineered T cell does notexpress B2M, wherein the engineered T cell does not express HLA-DP,HLA-DQ, and/or HLA-DR antigens, wherein the engineered T cell does notexpress CIITA, and/or wherein the engineered T cell does not expressTCR-alpha and TCR-beta.

In some embodiments, the engineered T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and/or the first exogenouspolynucleotide encoding CAR inserted into the TRAC locus, into the TRBlocus, into the B2M locus, or into the CIITA locus. In some embodiments,the exogenous polynucleotide is inserted into at least one allele of thecell using viral transduction, for example, with a vector. In someembodiments, the vector is a pseudotyped, self-inactivating lentiviralvector that carries the exogenous polynucleotide. In some embodiments,the vector is a self-inactivating lentiviral vector pseudotyped with avesicular stomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the non-activated T cell and/or the engineered Tcell of the present technology are in a subject. In other embodiments,the non-activated T cell and/or the engineered T cell of the presenttechnology are in vitro.

In some embodiments, the non-activated T cell and/or the engineered Tcell of the present technology express a CD8 binding agent. In someembodiments, the CD8 binding agent is an anti-CD8 antibody. In someembodiments, the anti-CD8 antibody is selected from the group consistingof a mouse anti-CD8 antibody, a rabbit anti-CD8 antibody, a humananti-CD8 antibody, a humanized anti-CD8 antibody, a camelid (e.g.,llama, alpaca, camel) anti-CD8 antibody, and a fragment thereof. In someembodiments, the fragment thereof is an scFV or a VHH. In someembodiments, the CD8 binding agent binds to a CD8 alpha chain and/or aCD8 beta chain.

In some embodiments, the CD8 binding agent is fused to a transmembranedomain incorporated in the viral envelope. In some embodiments, thelentivirus vector is pseudotyped with a viral fusion protein. In someembodiments, the viral fusion protein comprises one or moremodifications to reduce binding to its native receptor.

In some embodiments, the viral fusion protein is fused to the CD8binding agent. In some embodiments, the viral fusion protein comprisesNipah virus F glycoprotein and Nipah virus G glycoprotein fused to theCD8 binding agent. In some embodiments, the lentivirus vector does notcomprise a T cell activating molecule or a T cell costimulatorymolecule. In some embodiments, the lentivirus vector encodes the firstexogenous polynucleotide and/or the second exogenous polynucleotide.

In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using viral transduction, for example, witha vector. In some embodiments, the vector is a pseudotyped,self-inactivating lentiviral vector that carries the exogenouspolynucleotide. In some embodiments, the vector is a self-inactivatinglentiviral vector pseudotyped with a vesicular stomatitis VSV-Genvelope, and which carries the exogenous polynucleotide. In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction. In some embodiments, theexogenous polynucleotide is inserted into at least one allele of thecell using a lentivirus based viral vector.

In some embodiments, following transfer into a first subject, thenon-activated T cell or the engineered T cell exhibits one or moreresponses selected from the group consisting of (a) a T cell response,(b) an NK cell response, and (c) a macrophage response, that are reducedas compared to a wild-type cell following transfer into a secondsubject. In some embodiments, the first subject and the second subjectare different subjects. In some embodiments, the macrophage response isengulfment.

In some embodiments, following transfer into a subject, thenon-activated T cell or the engineered T cell exhibits one or moreselected from the group consisting of (a) reduced TH1 activation in thesubject, (b) reduced NK cell killing in the subject, and (c) reducedkilling by whole PBMCs in the subject, as compared to a wild-type cellfollowing transfer into the subject.

In some embodiments, following transfer into a subject, thenon-activated T cell or the engineered T cell elicits one or moreselected from the group consisting of (a) reduced donor specificantibodies in the subject, (b) reduced IgM or IgG antibodies in thesubject, and (c) reduced complement-dependent cytotoxicity (CDC) in asubject, as compared to a wild-type cell following transfer into thesubject.

In some embodiments, the non-activated T cell or the engineered T cellis transduced with a lentivirus vector comprising a CD8 binding agentwithin the subject. In some embodiments, the lentivirus vector carries agene encoding the CAR and/or CD47.

Provided herein are pharmaceutical compositions comprising a populationof the non-activated T cells and/or the engineered T cells of thepresent technology and a pharmaceutically acceptable additive, carrier,diluent or excipient.

Provided herein are methods comprising administering to a subject acomposition comprising a population of the non-activated T cells and/orthe engineered T cells of the present technology, or one or more thepharmaceutical compositions of the present technology.

In some embodiments, the subject is not administered a T cell activatingtreatment before, after, and/or concurrently with administration of thecomposition. In some embodiments, the T cell activating treatmentcomprises lymphodepletion.

Provided herein are methods of treating a subject suffering from cancer,comprising administering to a subject a composition comprising apopulation of the non-activated T cells and/or the engineered T cells ofthe present technology, or one or more the pharmaceutical compositionsof the present technology, wherein the subject is not administered a Tcell activating treatment before, after, and/or concurrently withadministration of the composition. In some embodiments, the T cellactivating treatment comprises lymphodepletion.

Provided herein are methods for expanding T cells capable of recognizingand killing tumor cells in a subject in need thereof within the subject,comprising administering to a subject a composition comprising apopulation of the non-activated T cells and/or the engineered T cells ofthe present technology, or one or more the pharmaceutical compositionsof the present technology, wherein the subject is not administered a Tcell activating treatment before, after, and/or concurrently withadministration of the composition. In some embodiments, the T cellactivating treatment comprises lymphodepletion.

Provided herein are dosage regimens for treating a disease or disorderin a subject comprising administration of a pharmaceutical compositioncomprising a population of the non-activated T cells and/or theengineered T cells of the present technology, or one or more thepharmaceutical compositions of the present technology, and apharmaceutically acceptable additive, carrier, diluent or excipient,wherein the pharmaceutical composition is administered in about 1-3doses.

Provided herein is an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type cell or a control cell, theengineered cell further comprising a set of exogenous polynucleotidescomprising a first exogenous polynucleotide encoding CD47 and a secondexogenous polynucleotide encoding a chimeric antigen receptor (CAR),wherein the first and/or second exogenous polynucleotides are insertedinto a specific locus of at least one allele of the cell.

In some embodiments, the specific locus is selected from the groupconsisting of a safe harbor or target locus, a B2M locus, a CIITA locus,a TRAC locus and a TRB locus.

In some embodiments, the first exogenous polynucleotide encoding CD47 isinserted into the specific locus selected from the group consisting of asafe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locusand a TRB locus.

In some embodiments, the second exogenous polynucleotide encoding theCAR is inserted into the specific locus selected from the groupconsisting of a safe harbor or target locus, a B2M locus, a CIITA locus,a TRAC locus and a TRB locus.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto different loci.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto the same locus.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto the B2M locus.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto the CIITA locus.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto the TRAC locus.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto the TRB locus.

In some embodiments, the first exogenous polynucleotide encoding CD47and the second exogenous polynucleotide encoding the CAR are insertedinto the safe harbor or target locus.

In some embodiments, the safe harbor locus is selected from the groupconsisting of a CCR5 gene locus, a PPP1R12C gene locus, a CLYBL genelocus, and a Rosa gene locus, and the target locus is selected from thegroup consisting of a CXCR4 gene locus, an albumin gene locus, a SHS231gene locus, an F3 (CD142) gene locus, a MICA gene locus, a MICB genelocus. a LRP1 (CD91) gene locus, a HMGB1 gene locus, an ABO gene locus,ad RHD gene locus, a FUT1 locus, and a KDM5D gene locus.

In some embodiments, the CAR is selected from the group consisting of aCD19-specific CAR and a CD22-specific CAR.

In some embodiments, the CD19-specific CAR is substantially equivalentto the CD19-specific CAR of any one of the CAR-T cell based therapiesselected from the group consisting of axicabtagene ciloleucel,lisocabtagene maraleucel, brexucabtagene autoleucel, andtisagenlecleucel.

In some embodiments, the CAR is a bispecific CAR.

In some embodiments, the CAR is a CD19/CD22-bispecific CAR.

In some embodiments, the engineered cell does not express HLA-A, HLA-B,and/or HLA-C antigens.

In some embodiments, the engineered cell does not express B2M.

In some embodiments, the engineered cell does not express HLA-DP,HLA-DQ, and/or HLA-DR antigens.

In some embodiments, the engineered cell does not express CIITA.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered celldoes not express TCR-alpha and/or TCR-beta.

In some embodiments, the engineered cell is a pluripotent stem cell.

In some embodiments, the engineered cell is an induced pluripotent stemcell.

In some embodiments, the engineered cell is a differentiated cellderived from an induced pluripotent stem cell.

In some embodiments, the differentiated cell is selected from the groupconsisting of an NK cell and a T cell.

In some embodiments, the engineered cell is a cell derived from aprimary T cell.

In some embodiments, the cell derived from the primary T cell is derivedfrom a pool of T cells comprising primary T cells from one or more donorsubjects who are different from a recipient subject.

In some embodiments, the engineered cell retains pluripotency and/orretains differentiation potential.

In some embodiments, following transfer into a first subject, theengineered cell exhibits one or more responses selected from the groupconsisting of (a) a T cell response, (b) an NK cell response, and (c) amacrophage response, that are reduced as compared to a wild-type cellfollowing transfer into a second subject.

In some embodiments, the first subject and the second subject aredifferent subjects.

In some embodiments, the macrophage response is engulfment.

In some embodiments, following transfer into a subject the engineeredcell exhibits one or more selected from the group consisting of (a)reduced TH1 activation in the subject, (b) reduced NK cell killing inthe subject, and (c) reduced killing by whole PBMCs in the subject, ascompared to a wild-type cell following transfer into the subject.

In some embodiments, following transfer into a subject the engineeredcell elicits one or more selected from the group consisting of (a)reduced donor specific antibodies in the subject, (b) reduced IgM or IgGantibodies in the subject, and (c) reduced complement-dependentcytotoxicity (CDC) in a subject, as compared to a wild-type cellfollowing transfer into the subject.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the TRAClocus.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into the TRAClocus.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the TRBlocus.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into the TRBlocus.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the B2Mlocus.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into a B2M locus.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising first exogenous polynucleotide encoding CD47 and/or thesecond exogenous polynucleotide encoding CAR inserted into the CIITAlocus.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding CAR inserted into a CIITAlocus.

Provided herein is an engineered cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type cell or a control cell.

In some embodiments, the engineered cell does not express HLA-A, HLA-Band/or HLA-C antigens.

In some embodiments, the engineered cell does not express CIITA.

In some embodiments, the engineered cell does not express HLA-DP,HLA-DQ, and/or HLA-DR antigens.

In some embodiments, the engineered cell does not express B2M.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered celldoes not express TCR-alpha.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered celldoes not express TCR-beta.

In some embodiments, the engineered cell overexpresses CD47 relative toa wild-type cell or a control cell.

In some embodiments, the engineered cell is a pluripotent stem cell.

In some embodiments, the engineered cell is an induced pluripotent stemcell.

In some embodiments, the engineered cell is a differentiated cellderived from an induced pluripotent stem cell.

In some embodiments, the differentiated cell is selected from the groupconsisting of an NK cell and a T cell.

In some embodiments, the engineered cell is a cell derived from aprimary T cell.

In some embodiments, the cell derived from the primary T cell is derivedfrom a pool of T cells comprising primary T cells from one or more donorsubjects who are different from a recipient subject.

In some embodiments, the engineered cell retains pluripotency and/orretains differentiation potential.

In some embodiments, following transfer into a first subject, theengineered cell exhibits one or more responses selected from the groupconsisting of (a) a T cell response, (b) an NK cell response, and (c) amacrophage response, that are reduced as compared to a wild-type cellfollowing transfer into a second subject.

In some embodiments, the first subject and the second subject aredifferent subjects.

In some embodiments, the macrophage response is engulfment.

In some embodiments, following transfer into a subject the engineeredcell exhibits one or more selected from the group consisting of (a)reduced TH1 activation in the subject, (b) reduced NK cell killing inthe subject, and (c) reduced killing by whole PBMCs in the subject, ascompared to a wild-type cell following transfer into the subject.

In some embodiments, following transfer into a subject the engineeredcell elicits one or more selected from the group consisting of (a)reduced donor-specific antibodies in the subject, (b) reduced IgM or IgGantibodies in the subject, and (c) reduced complement-dependentcytotoxicity (CDC) in the subject, as compared to a wild-type cellfollowing transfer into the subject.

In some embodiments, the engineered cell is selected from the groupconsisting a pluripotent stem cell, an induced pluripotent stem cell, aT cell differentiated from an induced pluripotent stem cell, a primary Tcell, and a cell derived from a primary T cell, and the engineered cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) and/orTRB^(indel/indel) cell.

In some embodiments, the engineered cell is a hypoimmunogenic cell.

In some embodiments, the wild type cell or the control cell is astarting material.

In some embodiments, the first and/or second exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.

In some embodiments, the viral transduction is via a lentivirus basedviral vector.

In some embodiments, the lentivirus based viral vector is a pseudotyped,self-inactivating lentiviral vector that carries the first and/or secondexogenous polynucleotides.

In some embodiments, the lentivirus based viral vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the first and/or secondexogenous polynucleotides.

Provided herein is a pharmaceutical composition comprising a populationof the engineered cells as described herein and a pharmaceuticallyacceptable additive, carrier, diluent or excipient.

In some embodiments, the pharmaceutically acceptable additive, carrier,diluent or excipient comprises one or more selected from the groupconsisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, humanserum albumin (HSA), dimethylsulfoxide (DMSO), and a combinationthereof.

In some embodiments of the composition, the composition furthercomprises a pharmaceutically acceptable buffer.

In some embodiments, the pharmaceutically acceptable buffer is neutralbuffer saline or phosphate buffered saline.

Provided herein is a pharmaceutical composition comprising a populationof the engineered cells as described herein, a base solution ofCryoStor® CSB at a concentration of about 70-80% w/w, and one or more ofabout 20-30% w/w PlasmaLyte-A™, about 0.3-5.3% w/v human serum albumin(HSA), about 0-20% v/v dimethylsulfoxide (DMSO), and about 100-400 mMtrehalose.

Provided herein is a pharmaceutical composition comprising a populationof the engineered cells as described herein, a base solution ofPlasmaLyte-A™ at a concentration of about 20-30% w/w, and one or more ofabout 70-80% w/w CryoStor® CSB, about 0.3-5.3% w/v human serum albumin(HSA), about 0-20% v/v dimethylsulfoxide (DMSO), and about 100-400 mMtrehalose.

Provided herein is a pharmaceutical composition comprising a populationof the engineered cells as described herein, about 0.3-5.3% w/v humanserum albumin (HSA), and one or more of about 70-80% w/w CryoStor® CSB,about 20-30% w/w PlasmaLyte-A™, about 0-20% v/v dimethylsulfoxide(DMSO), and about 100-400 mM trehalose.

Provided herein is a pharmaceutical composition comprising a populationof the engineered cells as described herein, about 0-20% v/vdimethylsulfoxide (DMSO), and one or more of about 70-80% w/w CryoStor®CSB, about 20-30% w/w PlasmaLyte-A™, about 0.3-5.3% w/v human serumalbumin (HSA), and about 100-400 mM trehalose.

Provided herein is a pharmaceutical composition comprising a populationof the engineered cells as described herein, about 100-400 mM trehalose,and one or more of about 70-80% w/w CryoStor® CSB, about 20-30% w/wPlasmaLyte-A™, about 0.3-5.3% w/v human serum albumin (HSA), and about0-20% v/v dimethylsulfoxide (DMSO).

In some embodiments, the pharmaceutical composition comprises about 75%w/w of CryoStor® CSB.

In some embodiments, the pharmaceutical composition comprises about 25%w/w of PlasmaLyte-A™.

In some embodiments, the pharmaceutical composition comprises about 0.3%w/v of HSA.

In some embodiments, the pharmaceutical composition comprises about 7.5%v/v of DMSO.

Provided herein is a pharmaceutical composition comprising a populationof the engineered cells as described herein, a base solution ofCryoStor® CSB at a concentration of about 75% w/w, about 25% w/wPlasmaLyte-A™, about 0.3% w/v human serum albumin (HSA), and about 7.5%v/v dimethylsulfoxide (DMSO).

In some embodiments, the population of the engineered cells is up toabout 8.0×10⁸ cells.

In some embodiments, the population of the engineered cells is up toabout 6.0×10⁸ cells.

In some embodiments, the population of the engineered cells is fromabout 1.0×10⁶ to about 2.5×10⁸ cells.

In some embodiments, the population of the engineered cells is fromabout 2.0×10⁶ to about 2.0×10⁸ cells.

In some embodiments, the population of the engineered cells ranges fromabout 5 ml to about 80 ml.

In some embodiments, the population of the engineered cells ranges fromabout 10 ml to about 70 ml.

In some embodiments, the population of the engineered cells ranges fromabout 10 ml to about 50 ml.

In some embodiments, the composition is formulated for administration ina single dose.

In some embodiments, the composition is formulated for administration inup to three doses.

In some embodiments, the composition is formulated for administration ofa single dose to a subject takes a duration of time of about 60 minutesor less.

In some embodiments, the composition is formulated for administration ofa single dose to a subject takes a duration of time of about 30 minutesor less.

In some embodiments, the population of engineered cells of thepharmaceutical composition or progeny thereof exhibit at least 40%survival in a subject after 10 days following administration.

In some embodiments, the population of engineered cells of thepharmaceutical composition or progeny thereof exhibit at least 80%survival in a subject after about 2 weeks following administration.

In some embodiments, the population of engineered cells of thepharmaceutical composition or progeny thereof exhibit at least 100%survival in a subject after about 3 weeks following administration.

In some embodiments, the population of engineered cells of thepharmaceutical composition or progeny thereof exhibit at least 150%survival in a subject after about 4 weeks following administration.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administration of a pharmaceutical compositioncomprising a population of engineered cells as described herein and apharmaceutically acceptable additive, carrier, diluent or excipient,wherein the pharmaceutical composition is administered in about 1-3doses.

In some embodiments, the pharmaceutical composition administered is upto about 6.0×10⁸ cells in about 1-3 doses.

In some embodiments, the pharmaceutical composition administered is fromabout 0.6×10⁶ to about 6.0×10⁸ cells in about 1-3 doses.

In some embodiments, the pharmaceutical composition administered is fromabout 0.2×10⁶ to about 5.0×10⁶ cells per kg of the subject's body weightin about 1-3 doses, if the subject has a body weight of 50 kg or less.

In some embodiments, the pharmaceutical composition administered is fromabout 0.1×10⁸ to about 2.5×10⁸ cells in about 1-3 doses, if the subjecthas a body weight greater than 50 kg.

In some embodiments, the pharmaceutical composition administered is fromabout 2.0×10⁶ cells per kg of the subject's body weight and up to about2.×10⁸ cells in about 1-3 doses.

In some embodiments, the administration of a single dose to the subjecttakes a duration of time of about 60 minutes or less.

In some embodiments, the administration of a single dose to the subjecttakes a duration of time of about 30 minutes or less.

In some embodiments, the pharmaceutically acceptable additive, carrier,diluent or excipient comprises one or more selected from the groupconsisting of Plasma-Lyte A©, dextrose, dextran, sodium chloride, humanserum albumin (HSA), dimethylsulfoxide (DMSO), and a combinationthereof.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable buffer.

In some embodiments, the pharmaceutically acceptable buffer is neutralbuffer saline or phosphate buffered saline.

In some embodiments, the administration of the pharmaceuticalcomposition, the population of cells or progeny thereof are present inthe subject up to 9 months.

In some embodiments, the administration of the pharmaceuticalcomposition, the population of cells or progeny thereof are present inthe subject at least 2 years or more.

In some embodiments, the administration of the pharmaceuticalcomposition, the population of engineered cells or progeny thereofexhibit at least 40% survival in a subject after about 10 days followingadministration.

In some embodiments, the administration of the pharmaceuticalcomposition, the population of engineered cells or progeny thereofexhibit at least 80% survival in a subject after about 2 weeks followingadministration.

In some embodiments, the administration of the pharmaceuticalcomposition, the population of engineered cells or progeny thereofexhibit at least 100% survival in a subject after about 3 weeksfollowing administration.

In some embodiments, the administration of the pharmaceuticalcomposition, the population of engineered cells or progeny thereofexhibit at least 150% survival in a subject after about 4 weeksfollowing administration.

In some embodiments, the administration of 2-3 doses to the subjectoccurs such that each dose is administered ranging from 1 to 24 hoursapart.

In some embodiments, the administration of 2-3 doses to the subjectoccurs such that each dose is administered ranging from 1 to 28 daysapart.

In some embodiments, the administration of 2-3 doses to the subjectoccurs such that each dose is administered ranging from 1 to 6 weeksapart.

In some embodiments, the administration of 2-3 doses to the subjectoccurs such that each dose is administered ranging from 1 to 12 monthsor more apart.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising

-   -   (i) an engineered cell comprising reduced expression of HLA-A,        HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,        and/or TCR-beta, the engineered cell further comprising a set of        exogenous polynucleotides encoding CD47 and a chimeric antigen        receptor (CAR), wherein the set of exogenous polynucleotides are        inserted into a safe harbor or target locus of at least one        allele of the cell; and    -   (ii) a pharmaceutically acceptable additive, carrier, diluent or        excipient,    -   wherein the pharmaceutical composition comprises up to about        6.0×10⁸ cells.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising

-   -   (i) an engineered cell comprising reduced expression of HLA-A,        HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,        and/or TCR-beta, the engineered cell further comprising a set of        exogenous polynucleotides encoding CD47 and a chimeric antigen        receptor (CAR), wherein the set of exogenous polynucleotides are        inserted into a safe harbor or target locus of at least one        allele of the cell; and    -   (ii) a pharmaceutically acceptable additive, carrier, diluent or        excipient,    -   wherein the pharmaceutical composition is administered in 1-3        doses.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising

-   -   (i) an engineered cell comprising reduced expression of HLA-A,        HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,        and/or TCR-beta, the engineered cell further comprising a set of        exogenous polynucleotides encoding CD47 and a chimeric antigen        receptor (CAR), wherein the set of exogenous polynucleotides are        inserted into a safe harbor or target locus of at least one        allele of the cell; and    -   (ii) a pharmaceutically acceptable additive, carrier, diluent or        excipient,    -   wherein a dose of the pharmaceutical composition is administered        for a duration of time of about 60 minutes or less.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising

-   -   (i) an engineered cell comprising reduced expression of HLA-A,        HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,        and/or TCR-beta; and    -   (ii) a pharmaceutically acceptable additive, carrier, diluent or        excipient,    -   wherein the pharmaceutical composition comprises up to about        6.0×10⁸ cells.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising

-   -   (i) an engineered cell comprising reduced expression of HLA-A,        HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,        and/or TCR-beta; and    -   (ii) a pharmaceutically acceptable additive, carrier, diluent or        excipient,    -   wherein the pharmaceutical composition is administered in 1-3        doses.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administering a pharmaceutical compositioncomprising

-   -   (i) an engineered cell comprising reduced expression of HLA-A,        HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,        and/or TCR-beta; and    -   (ii) a pharmaceutically acceptable additive, carrier, diluent or        excipient,    -   wherein a dose of the pharmaceutical composition is administered        for a duration of 60 minutes or less.

Provided herein is a method of treating a cancer in a subject comprisingadministration of the engineered cell as described herein, thepharmaceutical composition as described herein, or the dosage regimen asdescribed herein to the subject.

In some embodiments, the cancer is a CD19+ cancer.

Provided herein is a method of preventing T cell exhaustion in a subjectcomprising administration of the engineered cell as described herein tothe subject, wherein the CAR is a CD19-specific CAR or a CD22-specificCAR.

In some embodiments, the CD19-specific CAR is substantially equivalentto the CD19-specific CAR of any one of the CAR-T cell based therapiesselected from the group consisting of axicabtagene ciloleucel,lisocabtagene maraleucel, brexucabtagene autoleucel, andtisagenlecleucel.

Provided herein is a method of preventing T cell exhaustion or treatinga disease in a subject comprising:

-   -   (i) administration of a first dosage regimen comprising a first        population of engineered cells as described herein to the        subject at a first timepoint, and    -   (ii) administration of a second dosage regimen comprising a        second population of engineered cells as described herein to the        subject at a second timepoint,    -   wherein the first dosage regimen and the second dosage regimen        are different.

A method of preventing T cell exhaustion or treating a disease in asubject comprising:

-   -   (i) administration of a first dosage regimen comprising a first        population of engineered cells as described herein to the        subject at a first timepoint, and    -   (ii) administration of a second dosage regimen comprising a        second population of engineered cells as described herein to the        subject at a second timepoint,    -   wherein the first population of engineered cells and the second        population of engineered cells both comprise the same chimeric        antigen receptor.

Provided herein is a method of preventing T cell exhaustion or treatinga disease in a subject comprising:

-   -   (i) administration of a first dosage regimen comprising a first        population of engineered cells as described herein to the        subject at a first timepoint, and    -   (ii) administration of a second dosage regimen comprising a        second population of engineered cells as described herein to the        subject at a second timepoint,    -   wherein the first population of engineered cells and the second        population of engineered cells both comprise different chimeric        antigen receptors.

Provided herein is a method of preventing T cell exhaustion or treatinga disease in a subject comprising:

-   -   (i) administration of a first dosage regimen comprising a first        population of engineered cells as described herein to the        subject at a first timepoint, and    -   (ii) administration of a second dosage regimen comprising a        second population of engineered cells as described herein to the        subject at a second timepoint,    -   wherein the engineered cells of the first population comprise a        first chimeric antigen receptor that binds a first antigen and        the engineered cells of the second population comprise a second        chimeric antigen receptor that binds a second antigen, and        wherein the first antigen and the second antigen are the same.

Provided herein is a method of preventing T cell exhaustion or treatinga disease in a subject comprising:

-   -   (i) administration of a first dosage regimen comprising a first        population of engineered cells as described herein to the        subject at a first timepoint, and    -   (ii) administration of a second dosage regimen comprising a        second population of engineered cells as described herein to the        subject at a second timepoint,    -   wherein the engineered cells of the first population comprise a        first chimeric antigen receptor that binds a first antigen and        the engineered cells of the second population comprise a second        chimeric antigen receptor that binds a second antigen, and        wherein the first antigen and the second antigen are different.

In some embodiments, the engineered cell is not activated.

Provided herein is a non-activated T cell comprising reduced expressionof HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type T cell, and a first exogenouspolynucleotide encoding a chimeric antigen receptor (CAR).

In some embodiments, the non-activated T cell is a primary T cell.

In some embodiments, the non-activated T cell is differentiated from theengineered cell as described herein.

In some embodiments, the T cell is a CD8⁺ T cell.

In some embodiments, the non-activated T cell has not been treated withan anti-CD3 antibody, an anti-CD28 antibody, a T cell activatingcytokine, or a soluble T cell costimulatory molecule.

In some embodiments, the anti-CD3 antibody is OKT3.

In some embodiments, the anti-CD28 antibody is CD28.2.

In some embodiments, the T cell activating cytokine is selected from thegroup consisting of IL-2, IL-7, IL-15, and IL-21.

In some embodiments, the soluble T cell costimulatory molecule isselected from the group consisting of an anti-CD28 antibody, ananti-CD80 antibody, an anti-CD86 antibody, an anti-CD137L antibody, andan anti-ICOS-L antibody.

In some embodiments, the non-activated T cell does not expressactivation markers.

In some embodiments, the non-activated T cell expresses CD3 and CD28,and wherein the CD3 and/or CD28 are inactive.

In some embodiments, the first exogenous polynucleotide is carried by alentiviral vector that comprises a CD8 binding agent.

In some embodiments, the non-activated T cell further comprises a secondexogenous polynucleotide encoding CD47.

In some embodiments, the first and/or second exogenous polynucleotidesare inserted into a specific locus of at least one allele of the T cell.

In some embodiments, the specific locus is selected from the groupconsisting of a safe harbor or target locus, a B2M locus, a CIITA locus,a TRAC locus, and a TRB locus.

In some embodiments, the second exogenous polynucleotide encoding CD47is inserted into the specific locus selected from the group consistingof a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAClocus and a TRB locus.

In some embodiments, the first exogenous polynucleotide encoding the CARis inserted into the specific locus selected from the group consistingof a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAClocus and a TRB locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto different loci.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the same locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the B2M locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the CIITA locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto a TCR gene locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the TRAC locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the TRB locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the safe harbor or target locus.

In some embodiments, the safe harbor or target locus is selected fromthe group consisting of a CCR5 gene locus, a CXCR4 gene locus, aPPP1R12C gene locus, an albumin gene locus, a SHS231 gene locus, a CLYBLgene locus, a Rosa gene locus, an F3 (CD142) gene locus, a MICA genelocus, a MICB gene locus, a LRP1 (CD91) gene locus, a HMGB1 gene locus,an ABO gene locus, an RHD gene locus, a FUT1 locus, and a KDM5D genelocus.

In some embodiments, the CAR is selected from the group consisting of aCD19-specific CAR and a CD22-specific CAR.

In some embodiments, the CD19-specific CAR is substantially equivalentto the CD19-specific CAR of any one of the CAR-T cell based therapiesselected from the group consisting of axicabtagene ciloleucel,lisocabtagene maraleucel, brexucabtagene autoleucel, andtisagenlecleucel.

In some embodiments, the CAR is a bispecific CAR.

In some embodiments, the bispecific CAR is a CD19/CD22 bispecific CAR.

In some embodiments, the non-activated T cell does not express HLA-A,HLA-B, and/or HLA-C antigens.

In some embodiments, the non-activated T cell does not express B2M.

In some embodiments, the non-activated T cell does not express HLA-DP,HLA-DQ, and/or HLA-DR antigens.

In some embodiments, the non-activated T cell does not express CIITA.

In some embodiments, the non-activated T cell does not express TCR-alphaand/or TCR-beta.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the TRAC locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into the TRAC locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the TRB locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into the TRB locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and/or the first exogenouspolynucleotide encoding CAR inserted into the B2M locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into a B2M locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the CIITA locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into a CIITA locus.

Provided herein is an engineered T cell comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type T cell, wherein the engineered Tcell further comprises a first exogenous polynucleotide encoding achimeric antigen receptor (CAR) carried by a lentiviral vectorcomprising a CD8 binding agent.

In some embodiments, the engineered T cell is a primary T cell.

In some embodiments, the engineered T cell is differentiated from theengineered cell as described herein.

In some embodiments, the T cell is a CD8⁺ T cell.

In some embodiments, the engineered T cell has not been treated with ananti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine,or a soluble T cell costimulatory molecule.

In some embodiments, the anti-CD3 antibody is OKT3, wherein theanti-CD28 antibody is CD28.2, wherein the T cell activating cytokine isselected from the group consisting of IL-2, IL-7, IL-15, and IL-21, andwherein the soluble T cell costimulatory molecule is selected from thegroup consisting of an anti-CD28 antibody, an anti-CD80 antibody, ananti-CD86 antibody, an anti-CD137L antibody, and an anti-ICOS-Lantibody.

In some embodiments, the engineered T cell does not express activationmarkers.

In some embodiments, the engineered T cell expresses CD3 and CD28, andwherein the CD3 and/or CD28 are inactive.

In some embodiments, the engineered T cell further comprises a secondexogenous polynucleotide encoding CD47.

In some embodiments, the first and/or second exogenous polynucleotidesare inserted into a specific locus of at least one allele of the T cell.

In some embodiments, the specific locus is selected from the groupconsisting of a safe harbor or target locus, a B2M locus, a CIITA locus,a TRAC locus, and a TRB locus.

In some embodiments, the second exogenous polynucleotide encoding CD47is inserted into the specific locus selected from the group consistingof a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAClocus and a TRB locus.

In some embodiments, the first exogenous polynucleotide encoding the CARis inserted into the specific locus selected from the group consistingof a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAClocus and a TRB locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto different loci.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the same locus.

In some embodiments, the second exogenous polynucleotide encoding CD47and the first exogenous polynucleotide encoding the CAR are insertedinto the B2M locus, the CIITA locus, the TRAC locus, the TRB locus, orthe safe harbor or target locus.

In some embodiments, the safe harbor or target locus is selected fromthe group consisting of a CCR5 gene locus, a CXCR4 gene locus, aPPP1R12C gene locus, an albumin gene locus, a SHS231 gene locus, a CLYBLgene locus, a Rosa gene locus, an F3 (CD142) gene locus, a MICA genelocus, a MICB gene locus, a LRP1 (CD91) gene locus, a HMGB1 gene locus,an ABO gene locus, an RHD gene locus, a FUT1 locus, and a KDM5D genelocus.

In some embodiments, the CAR is selected from the group consisting of aCD19-specific CAR and a CD22-specific CAR.

In some embodiments, the CD19-specific CAR is substantially equivalentto the CD19-specific CAR of any one of the CAR-T cell based therapiesselected from the group consisting of axicabtagene ciloleucel,lisocabtagene maraleucel, brexucabtagene autoleucel, andtisagenlecleucel.

In some embodiments, the engineered T cell does not express HLA-A,HLA-B, and/or HLA-C antigens, wherein the engineered T cell does notexpress B2M, wherein the engineered T cell does not express HLA-DP,HLA-DQ, and/or HLA-DR antigens, wherein the engineered T cell does notexpress CIITA, and/or wherein the engineered T cell does not expressTCR-alpha and/or TCR-beta.

In some embodiments, the engineered T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and/or the first exogenouspolynucleotide encoding CAR inserted into the TRAC locus, into the TRBlocus, into the B2M locus, or into the CIITA locus.

In some embodiments, the non-activated T cell is as described herein orthe engineered T cell is as described herein, wherein the non-activatedT cell or the engineered T cell is in a subject.

In some embodiments, the non-activated T cell is as described herein orthe engineered T cell is as described herein, wherein the non-activatedT cell or the engineered T cell is in vitro.

In some embodiments, the non-activated T cell is as described herein orthe engineered T cell is as described herein, wherein the CD8 bindingagent is an anti-CD8 antibody.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the anti-CD8 antibody is selected fromthe group consisting of a mouse anti-CD8 antibody, a rabbit anti-CD8antibody, a human anti-CD8 antibody, a humanized anti-CD8 antibody, acamelid anti-CD8 antibody, and a fragment thereof.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the fragment thereof is an scFV or aVHH.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the CD8 binding agent binds to a CD8alpha chain and/or a CD8 beta chain.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the CD8 binding agent is fused to atransmembrane domain incorporated in a viral envelope.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the lentivirus vector is pseudotypedwith a viral fusion protein.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the viral fusion protein comprises oneor more modifications to reduce binding to its native receptor.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the viral fusion protein is fused to theCD8 binding agent.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the viral fusion protein comprises Nipahvirus F glycoprotein and Nipah virus G glycoprotein fused to the CD8binding agent.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the lentivirus vector does not comprisea T cell activating molecule or a T cell costimulatory molecule.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the lentivirus vector encodes the firstexogenous polynucleotide and/or the second exogenous polynucleotide.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein, following transfer into a firstsubject, the non-activated T cell or the engineered T cell exhibits oneor more responses selected from the group consisting of (a) a T cellresponse, (b) an NK cell response, and (c) a macrophage response, thatare reduced as compared to a wild-type cell following transfer into asecond subject.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the first subject and the second subjectare different subjects.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the macrophage response is engulfment.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein, following transfer into a subject, thenon-activated T cell or the engineered T cell exhibits one or moreselected from the group consisting of (a) reduced TH1 activation in thesubject, (b) reduced NK cell killing in the subject, and (c) reducedkilling by whole PBMCs in the subject, as compared to a wild-type cellfollowing transfer into the subject.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein, following transfer into a subject, thenon-activated T cell or the engineered T cell elicits one or moreselected from the group consisting of (a) reduced donor specificantibodies in the subject, (b) reduced IgM or IgG antibodies in thesubject, and (c) reduced complement-dependent cytotoxicity (CDC) in asubject, as compared to a wild-type cell following transfer into thesubject.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the non-activated T cell or theengineered T cell is transduced with a lentivirus vector comprising aCD8 binding agent within the subject.

In some embodiments, the non-activated T cell or the engineered T cellis as described herein, wherein the lentivirus vector carries a geneencoding the CAR and/or CD47.

Provided herein is a pharmaceutical composition comprising a populationof the non-activated T cell or the engineered T cell is as describedherein and a pharmaceutically acceptable additive, carrier, diluent orexcipient.

Provided herein is a method comprising administering to a subject acomposition comprising the non-activated T cell as described herein, theengineered T cell as described herein, or the pharmaceutical compositionas described herein.

In some embodiments, the subject is not administered a T cell activatingtreatment before, after, and/or concurrently with the administration ofthe composition.

In some embodiments, the T cell activating treatment compriseslymphodepletion.

Provided herein is a method of treating a subject suffering from cancer,comprising administering to a subject a composition comprising thenon-activated T cell as described herein, the engineered T cell asdescribed herein, or the pharmaceutical composition as described herein,wherein the subject is not administered a T cell activating treatmentbefore, after, and/or concurrently with the administration of thecomposition.

In some embodiments, the T cell activating treatment compriseslymphodepletion.

A method for expanding T cells capable of recognizing and killing tumorcells in a subject in need thereof within the subject, comprisingadministering to a subject a composition comprising the non-activated Tcell as described herein, the engineered T cell as described herein, orthe pharmaceutical composition as described herein, wherein the subjectis not administered a T cell activating treatment before, after, and/orconcurrently with the administration of the composition.

In some embodiments, the T cell activating treatment compriseslymphodepletion.

Provided herein is a dosage regimen for treating a disease or disorderin a subject comprising administration of a pharmaceutical compositioncomprising a population of subject a composition comprising thenon-activated T cell as described herein, and/or the engineered T cellas described herein, and a pharmaceutically acceptable additive,carrier, diluent or excipient, wherein the pharmaceutical composition isadministered in about 1-3 doses.

The present disclosure is related to U.S. Provisional Application filedon Dec. 31, 2020 (Attorney Docket No. 112864-5057-PR) and U.S.Provisional Application filed on Jan. 11, 2021 filed by Morrison andFoerester having Attorney Docket No. 18615-30046.00, the contents ofwhich are hereby incorporated by reference in their entirety. Detaileddescriptions of engineered and/or hypoimmunogenic cells, methods ofproducing thereof, and methods of using thereof are found in U.S.Provisional Application No. 63/065,342 filed on Aug. 13, 2020,WO2016/183041 filed May 9, 2015, WO2018/132783 filed Jan. 14, 2018,WO2020/018615 filed Jul. 17, 2019, WO2020/018620 filed Jul. 17, 2019,WO2020/168317 filed Feb. 16, 2020, the disclosures of which includingthe examples, sequence listings and figures are incorporated herein byreference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the characterization of hypoimmunogenic T cells describedherein. Such cells are HLA-I and HLA-II knock-out and CD47 knock-incells.

FIG. 2 shows the absence of NK cell mediated killing of thehypoimmunogenic T cells. In contrast, blocking CD47 with an anti-CD47antibody resulted in NK cell mediated killing of the cells. Mock T cellswere not killed by allogeneic NK cells (as expected). T cells lackingHLA-I/II were killed by NK cells. HLA-I/II knockout and CD47 knock-incells were not killed by NK cells. Blocking CD47 with magrolimab (e.g.,an anti-CD47 antibody) resulted in killing of HLA-I/II knockout, CD47knock-in cells, and thus highlights the protection by CD47.

FIG. 3 shows the absence of macrophage mediated killing of thehypoimmunogenic T cells. Control T cells were not killed by allogeneicmacrophages and T cells lacking HLA-I/II were killed by macrophages.HLA-I/II knockout and CD47 knock-in cells were not killed bymacrophages. Blocking CD47 with magrolimab (e.g., an anti-CD47 antibody)resulted in killing of HLA-I/II knockout, CD47 knock-in cells, and thushighlights the protection by CD47.

FIG. 4 shows the expression of the CD19-specific CAR and CD47 constructsin exemplary hypoimmunogenic CAR-T cells. The CD19-specific CAR-CD47 Tcells expressed exogenous CD47 at high levels. As used herein, the term“CD19-specific CAR-CD47 T cell” refers to a T cell exogenouslyexpressing a CD19-specific CAR and CD47.

FIGS. 5A and 5B depict killing of CD19+ tumor cells by exemplaryhypoimmunogenic CAR-T cells (CD19-specific CAR-CD47 T cells) in vitro ina dose-dependent manner. CD47 overexpression seemed not to affectCD19-specific CAR activity. CD19-specific CAR-CD47 T cells showedsimilar killing as control CD19-specific CAR-T cells (“CAR low” (FIG.5A) and “CAR high” (FIG. 5B) cells).

FIGS. 6A and 6B depict flow data confirming real-time cell analysis datashowing killing of CD19+ tumor cells by exemplary hypoimmunogenic CAR-Tcells (e.g., CD19-specific CAR-CD47 T cells) (“CAR low” (FIG. 6A) and“CAR high” (FIG. 6B) cells) in vitro in a dose-dependent manner. CD47overexpression did not after CD19-specific CAR activity in in vitroassays.

FIG. 7 shows killing of CD19+ tumor cells by exemplary hypoimmunogenicCAR-T cells (CD19-specific CAR-CD47 T cells) that were FACS sorted. Thetarget cell:effector cell ratio was 1:3 and the killing was analyzedusing a real-time, quantitative microelectronic biosensor system forcell analysis (xCELLigence® RTCA system, Agilent) for 48 hours and byflow cytometry. The data shows that CD19-specific CAR-T cells withendogenous CD47 expression killed the tumor cells. Also, CD19-specificCAR-T cells with exogenous CD47 expression killed the tumor cells.Control T cells (mock T cells) did not kill the tumor cells. CD47overexpression does not appear to affect CD19-specific CAR activity inin vitro assays.

FIG. 8 shows the growth of the cells described herein, in particular,the CD47-dependent and CD47-independent growth of CD19-specific CAR-Tcells and CD19-specific CAR-CD47 T cells.

FIG. 9A-B show the efficacy of the exemplary hypoimmunogenic CAR-T cellsin a mouse model with human CD19+ tumors. The whole animal scans showthe effects of CD19-specific CAR-T cells, CD19-specific CAR-CD47 Tcells, and mock T cells on CD19+ tumor cells.

FIG. 10 shows the efficacy of the exemplary hypoimmunogenic CAR-T cellsat various effector to Nalm6 target ratios.

FIG. 11 depicts cell viability and the frequency of TRAC, B2M and CIITAtriple knockouts in hypoimmunogenic CD19-specific CAR-CD47 T cells andcontrol T cells (CD19-specific CAR-EGFRt T cells and mock T cells) fourdays after nucleofection to introduce a CRISPR/Cas9 based gene editingsystem into the CD19-specific CAR-T cells and CD19-specific CAR-CD47 Tcells.

FIGS. 12A and 12B show the frequency (FIG. 12A) and MFI (FIG. 12B) of anexemplary CD19-specific CAR in hypoimmunogenic CD19-specific CAR-CD47 Tcells and control T cells (CD19-specific CAR-EGFRt T cells,tisagenlecleucel biosimilar/surrogate cells, and mock T cells).

FIG. 13 shows the frequency of both an exemplary CD19-specific CAR andCD47 molecules in hypoimmunogenic CD19-specific CAR-CD47 T cells andcontrol T cells (CD19-specific CAR-EGFRt T cells, tisagenlecleucelbiosimilar/surrogate cells, CD47 expressing T cells, and mock T cells).

FIG. 14 depicts the vector copy number in hypoimmunogenic CD19-specificCAR-CD47 T cells and control T cells (CD19-specific CAR-EGFRt T cells,tisagenlecleucel biosimilar/surrogate cells, and mock T cells) on day 8post-activation.

FIG. 15 depicts the expression of CD47 molecules in hypoimmunogenicCD19-specific CAR-CD47 T cells using a method for flow cytometricestimation of antibodies per cell (e.g., QuantiBRITE™, BD Biosciences).In this assay, exogenous CD47 expression is above 200,000 molecules percell.

FIGS. 16A-D, 17A-D-18A-C show that the presence of the triple geneinactivation of the TRAC, B2M and CIITA genes and overexpression of CD47proteins did not affect activity of an exemplary CD19-specific CAR inthe hypoimmunogenic CD19-specific CAR-CD47 T cells described herein.

FIGS. 19-22 provide the efficacy of the hypoimmunogenic CD19-specificCAR-CD47 T cells described herein in a mouse model with CD19+ tumorcells. Whole animal scans show killing of tumor cells by suchhypoimmunogenic CD19-specific CAR-T cells. The killing activity appearedto be in a dose-dependent manner.

FIG. 23 shows the efficacy of the hypoimmunogenic CD19-specific CAR-CD47T cells at varying effector: tumor cell ratios over a range of 0 to 28days.

FIG. 24 shows whole animal scans depicting killing of tumor cells byhypoimmunogenic CD19-specific CAR-CD47 T cells over range of 27 days.The effector:tumor cell ratio used was 7:1.

FIG. 25 shows exemplary test cells and control cells used in the study.Exemplary test cells include hypoimmunogenic CD19-specific CAR-CD47 Tcells that harbor genome edits of the B2M, CIITA and TRAC genes andoverexpress CD47 molecules and CD19-specific chimeric antigen receptors.Control cells include immunogenic CD19-specific CAR-T cellsco-expressing CD47 and EGFR as well as a tisagenlecleucel biosimilar orsurrogate.

FIG. 26 and FIG. 27 depict FACS analysis of the exemplaryhypoimmunogenic CD19-specific CAR-CD47 T cells and the absence of CD3,B2M, HLA-DR/HLA-DP/HLA-DQ, and HLA-A/HLA-B/HLA-C expression.

FIG. 28 shows FACS analysis of the expression of CD47 and CD19-CAR inhypoimmunogenic CD19-specific CAR-CD47 T cells.

FIG. 29 depicts the expression of CD3, B2M, TRAC, HLA-DR/HLA-DP/HLA-DQ,and HLA-A/HLA-B/HLA-C as determined by FACS and ICE in hypoimmunogenicCD19-specific CAR-CD47 T cells, CD19-specific CAR-EGFRt T cells, andmock T cells.

FIG. 30 shows that hypoimmunogenic CD19-specific CAR-CD47 T cells wereable to kill tumor cells equivalently to control CD19-specific CAR-Tcells in vitro. B cell leukemia killing kinetics and B cell leukemiatotal killing are depicted.

FIG. 31 provides a schematic diagram of the experimental approach ofExample 3 for studying cytokine-independent proliferation of TRAC, B2M,and CIITA Triple Knockout CAR-T Cells expressing a CD47 transgene(B2M^(−/−), CIITA^(−/−), TRAC^(−/−), CD47tg CD19-specific CAR-T cells,which are also known as tKO/CD47 CAR-T cells or as HIP CD19-CAR-Tcells).

FIG. 32A and FIG. 32B depict graphs that illustrate the proliferation ofthe B2M^(−/−), CIITA^(−/−), TRAC^(−/−), CD47tg CD19-specific CAR-T cells(tKO/CD47 CAR-T cells or HIP CD19-CAR-T cells) cultured either in mediasupplemented with IL-2 or without supplemented IL-2.

FIG. 33 provides a schematic diagram of the experimental approach ofExample 4.

FIG. 34 shows in vivo bioluminescent images to detect Nalm6-luc tumorcells in xenografted mice. The mice were administered with eithertKO/CD47 CAR-T cells (HIP CD19-CAR-T cells), control CAR-T cells(CD19-CAR-T cells), unedited T cells, or saline. The bioluminescenceimages show tumor progression at day 35, day 42, day 49 and day 56 ofthe study.

FIG. 35 provides a graph representing Nalm6-luc tumor cells detected inthe xenografted mice. Images of Nalm6-luc bearing mice show delayedtumor growth in tKO/CD47 CAR-T cells (HIP CD19-CAR-T cells) treated micewhen compared with control CAR-T (CD19-CAR-T) treated mice, unedited Tcell-treated mice and saline-treated mice.

FIG. 36 shows a schematic diagram of an experimental approach forgenerating B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel),CD47tg T cells expressing a CD19-specific CAR, also referred to astKO/CD47 CAR-T cells or HIP CD19-CAR-T cells. Such cells are CD47-CARlentivirus transduced and gene edited T cells (bottom row; HIP CAR-Tcells). The diagram also shows the generation of two types of controlcells: untransduced and unedited cells (top row; unedited T cells) andCAR-EGFRt lentivirus transduced and unedited cells (middle row; controlCAR-T cells).

FIG. 37 shows a flow chart of an illustrative serial positiveimmunomagnetic cell selection strategy for isolating CD8 T cells and CD4T cells from an enriched leukapheresis product collected from normal,healthy peripheral blood.

FIG. 38 provides a table of flow cytometry data that characterizes theHIP CAR-T cells (HIP CD19-CAR-T cells), control CAR-T cells (CD19-CAR-Tcells), and unedited T cells produced according to the method outlinedin FIG. 36.

FIG. 39A-D show the experimental results of Example 6. FIGS. 39A-B showElispot analysis of the Th1 (IFNg) response, and FIGS. 39C-D show theresults of the killing assay using CD19-CAR-T cells (FIG. 39C) or HIPCD19-CAR-T cells (FIG. 39D).

FIG. 40A and FIG. 40B show the viability and killing potency of HIPcells formulated in exemplary formulations described in Table 20. FIG.40A shows the pre-freeze (empty markers) or post-thaw (solid markers)viability of HIP cells prepared in the formulations, and FIG. 40B showsthe NALM-6 killing potency of the HIP cells prepared in theformulations.

FIGS. 41A-D show Th1 (IFNg) response as determined by Elispot forunsorted and sorted T cells.

FIGS. 42A-J show the quantification of tumor burden over time for thestudy described in Example 9.

FIGS. 43A-H shows the HIP CD19-CAR-T cell frequencies and expression ofCD47 in blood for the study described in Example 9. Specifically, FIGS.43A-F shows the frequency of HIP CD19-CAR-T cells in blood assessed atinterim bleeds and time of sacrifice, and FIG. 43G shows the CD47MFI ofCD19-CAR-T cells in blood at Day 108 in groups rechallenged with 5×10⁶HIP CD19-CAR-T cells and tKO CD19-CAR-T (HIP CD19-CAR-T) treated groups.One-way ANOVA with Tukey's multiple comparisons test performed on CAR+cell frequency data and two-way ANOVA with Bonferroni's multiplecomparisons test performed on blood CD47MFI data.

FIG. 44 shows exploratory autonomous growth assay cell count results forthe study described in Example 10.

Other objects, advantages and embodiments of the present disclosure willbe apparent from the detailed description following.

DETAILED DESCRIPTION I. Introduction

Described herein are engineered or modified immune evasive cells based,in part, on the hypoimmune editing platform described in WO2018132783,including but not limited to human immune evasive cells. To overcome theproblem of a subject's immune rejection of these primary and/or stemcell-derived transplants, the inventors have developed and describeherein hypoimmunogenic cells (e.g., hypoimmunogenic pluripotent cells,differentiated cells derived from such, and primary cells) thatrepresent a viable source for any transplantable cell type. Such cellsare protected from adaptive and/or innate immune rejection uponadministration to a recipient subject. Advantageously, the cellsdisclosed herein are not rejected by the recipient subject's immunesystem, regardless of the subject's genetic make-up, as they areprotected from adaptive and innate immune rejection upon administrationto a recipient subject. In some embodiments, the engineered and/orhypoimmunogenic cells do not express major histocompatibility complex(MHC) class I and class II antigens and/or T-cell receptors. In certainembodiments, the engineered and/or hypoimmunogenic cells do not expressMHC I and II antigens and/or T-cell receptors and overexpress CD47proteins. In certain embodiments, the engineered and/or hypoimmunogeniccells such as engineered and/or hypoimmunogenic T cells do not expressMHC I and II antigens and/or T-cell receptors, overexpress CD47 proteinsand express exogenous CARs.

In some embodiments, hypoimmunogenic cells outlined herein are notsubject to an innate immune cell rejection. In some instances,hypoimmunogenic cells are not susceptible to NK cell-mediated lysis. Insome instances, hypoimmunogenic cells are not susceptible to macrophageengulfment. In some embodiments, hypoimmunogenic cells are useful as asource of universally compatible cells or tissues (e.g., universal donorcells or tissues) that are transplanted into a recipient subject withlittle to no immunosuppressant agent needed. Such hypoimmunogenic cellsretain cell-specific characteristics and features upon transplantation,including, e.g., pluripotency, as well as being capable of engraftmentand functioning similarly to a corresponding native cell.

The technology disclosed herein utilizes expression of tolerogenicfactors and modulation (e.g., reduction or elimination) of MHC I, MHCII, and/or TCR expression in human cells. In some embodiments, genomeediting technologies utilizing rare-cutting endonucleases (e.g., theCRISPR/Cas, TALEN, zinc finger nuclease, meganuclease, and homingendonuclease systems) are also used to reduce or eliminate expression ofgenes involved in an immune response (e.g., by deleting genomic DNA ofgenes involved in an immune response or by insertions of genomic DNAinto such genes, such that gene expression is impacted) in the cells. Insome embodiments, genome editing technologies or other gene modulationtechnologies are used to insert tolerance-inducing (tolerogenic) factorsin human cells, rendering the cells and their progeny (include anydifferentiated cells prepared therefrom) able to evade immunerecognition upon engrafting into a recipient subject. As such, the cellsdescribed herein exhibit modulated expression of one or more genes andfactors that affect MHC I, MHC II, and/or TCR expression and evade therecipient subject's immune system.

The genome editing techniques enable double-strand DNA breaks at desiredlocus sites. These controlled double-strand breaks promote homologousrecombination at the specific locus sites. This process focuses ontargeting specific sequences of nucleic acid molecules, such aschromosomes, with endonucleases that recognize and bind to the sequencesand induce a double-stranded break in the nucleic acid molecule. Thedouble-strand break is repaired either by an error-prone non-homologousend-joining (NHEJ) or by homologous recombination (HR).

The practice of the numerous embodiments will employ, unless indicatedspecifically to the contrary, conventional methods of chemistry,biochemistry, organic chemistry, molecular biology, microbiology,recombinant DNA techniques, genetics, immunology, and cell biology thatare within the skill of the art, many of which are described below forthe purpose of illustration. Such techniques are explained fully in theliterature. See, e.g., Sambrook, et al., Molecular Cloning: A LaboratoryManual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual (2nd Edition, 1989); Maniatis et al., MolecularCloning: A Laboratory Manual (1982); Ausubel et al., Current Protocolsin Molecular Biology (John Wiley and Sons, updated July 2008); ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I &II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis ofComplex Genomes, (Academic Press, New York, 1992); Transcription andTranslation (B. Hames & S. Higgins, Eds., 1984); Perbal, A PracticalGuide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) CurrentProtocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies,E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology;as well as monographs in journals such as Advances in Immunology.

II. Definitions

As described in the present disclosure, the following terms will beemployed, and are defined as indicated below.

The term “autoimmune disease” refers to any disease or disorder in whichthe subject mounts an immune response against its own tissues and/orcells. Autoimmune disorders can affect almost every organ system in thesubject (e.g., human), including, but not limited to, diseases of thenervous, gastrointestinal, and endocrine systems, as well as skin andother connective tissues, eyes, blood and blood vessels. Examples ofautoimmune diseases include, but are not limited to Hashimoto'sthyroiditis, Systemic lupus erythematosus, Sjogren's syndrome, Graves'disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis,Myasthenia gravis and Diabetes.

The term “cancer” as used herein is defined as a hyperproliferation ofcells whose unique trait (e.g., loss of normal controls) results inunregulated growth, lack of differentiation, local tissue invasion, andmetastasis. With respect to the inventive methods, the cancer can be anycancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, braincancer, breast cancer, cancer of the anus, anal canal, or anorectum,cancer of the eye, cancer of the intrahepatic bile duct, cancer of thejoints, cancer of the neck, gallbladder, or pleura, cancer of the nose,nasal cavity, or middle ear, cancer of the oral cavity, cancer of thevulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, fibrosarcoma,gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer,kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer,lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovariancancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer,pharynx cancer, prostate cancer, rectal cancer, renal cancer, skincancer, small intestine cancer, soft tissue cancer, solid tumors,stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and/orurinary bladder cancer. As used herein, the term “tumor” refers to anabnormal growth of cells or tissues of the malignant type, unlessotherwise specifically indicated and does not include a benign typetissue.

The term “chronic infectious disease” refers to a disease caused by aninfectious agent wherein the infection has persisted. Such a disease mayinclude hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HSV-6,HSV-II, CMV, and EBV), and HIV/AIDS. Non-viral examples may includechronic fungal diseases such Aspergillosis, Candidiasis,Coccidioidomycosis, and diseases associated with Cryptococcus andHistoplasmosis. None limiting examples of chronic bacterial infectiousagents may be Chlamydia pneumoniae, Listeria monocytogenes, andMycobacterium tuberculosis. In some embodiments, the disorder is humanimmunodeficiency virus (HIV) infection. In some embodiments, thedisorder is acquired immunodeficiency syndrome (AIDS).

As used herein, “clinically effective amount” refers to an amountsufficient to provide a clinical benefit in the treatment and/ormanagement of a disease, disorder, or condition. In some embodiments, aclinically effective amount is an amount that has been shown to produceat least one improved clinical endpoint to the standard of care for thedisease, disorder, or condition. In some embodiments, a clinicallyeffective amount is an amount that has been demonstrated, for example ina clinical trial, to be sufficient to provide statistically significantand meaningful effectiveness for treating the disease, disorder, orcondition. In some embodiments, the clinically effective amount is alsoa therapeutically effective amount. In other embodiments, the clinicallyeffective amount is not a therapeutically effective amount.

In some embodiments, an alteration or modification (including, forexample, genetic alterations or modifications) described herein resultsin reduced expression of a target or selected polynucleotide sequence.In some embodiments, an alteration or modification described hereinresults in reduced expression of a target or selected polypeptidesequence. In some embodiments, an alteration or modification describedherein results in increased expression of a target or selectedpolynucleotide sequence. In some embodiments, an alteration ormodification described herein results in increased expression of atarget or selected polypeptide sequence.

In additional or alternative embodiments, the present disclosurecontemplates altering target polynucleotide sequences in any mannerwhich is available to the skilled artisan, e.g., utilizing a TALENsystem or RNA-guided transposases. It should be understood that althoughexamples of methods utilizing CRISPR/Cas (e.g., Cas9 and Cas12a) andTALEN are described in detail herein, the present disclosure is notlimited to the use of these methods/systems. Other methods of targeting,e.g., B2M, to reduce or ablate expression in target cells known to theskilled artisan can be utilized herein.

The terms “decrease,” “reduced,” “reduction,” and “decrease” are allused herein generally to mean a decrease by a statistically significantamount. However, for avoidance of doubt, decrease,” “reduced,”“reduction,” “decrease” means a decrease by at least 10% as compared toa reference level, for example a decrease by at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% decrease (i.e. absentlevel as compared to a reference sample), or any decrease between10-100% as compared to a reference level. In some embodiments, the cellsare engineered to have reduced expression of one or more targetsrelative to an unaltered or unmodified wild-type cell.

In some embodiments, the engineered and hypoimmunogenic cells describedare derived from an iPSC or a progeny thereof. As used herein, the term“derived from an iPSC or a progeny thereof” encompasses the initial iPSCthat is generated and any subsequent progeny thereof. As used herein,the term “progeny” encompasses, e.g., a first-generation progeny, i.e.,the progeny is directly derived from, obtained from, obtainable from orderivable from the initial iPSC by, e.g., traditional propagationmethods. The term “progeny” also encompasses further generations such assecond, third, fourth, fifth, sixth, seventh, or more generations, i.e.,generations of cells which are derived from, obtained from, obtainablefrom or derivable from the former generation by, e.g., traditionalpropagation methods. The term “progeny” also encompasses modified cellsthat result from the modification or alteration of the initial iPSC or aprogeny thereof.

The term “donor subject” refers to an animal, for example, a human fromwhom cells can be obtained. The “non-human animals” and “non-humanmammals” as used interchangeably herein, includes mammals such as rats,mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.The term “donor subject” also encompasses any vertebrate including butnot limited to mammals, reptiles, amphibians and fish. However,advantageously, the donor subject is a mammal such as a human, or othermammals such as a domesticated mammal, e.g., dog, cat, horse, and thelike, or production mammal, e.g., cow, sheep, pig, and the like. A“donor subject” can also refer to more than one donor, for example oneor more humans or non-human animals or non-human mammals.

The term “endogenous” refers to a referenced molecule or polypeptidethat is naturally present in the cell. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid naturally contained within the cell and notexogenously introduced. Similarly, the term when used in reference to apromoter sequence refers to a promoter sequence naturally containedwithin the cell and not exogenously introduced.

The term “engineered cell” as used herein refers to a cell that has beenaltered in at least some way by human intervention, including, forexample, by genetic alterations or modifications such that theengineered cell differs from a wild-type cell.

As used herein, the term “exogenous” in the context of a polynucleotideor polypeptide being expressed is intended to mean that the referencedmolecule or the referenced polypeptide is introduced into the cell ofinterest. The polypeptide can be introduced, for example, byintroduction of an encoding nucleic acid into the genetic material ofthe cells such as by integration into a chromosome or as non-chromosomalgenetic material such as a plasmid or expression vector. Therefore, theterm as it is used in reference to expression of an encoding nucleicacid refers to introduction of the encoding nucleic acid in anexpressible form into the cell. An exogenous polynucleotide can beinserted into at least one allele of the cell using viral transduction,for example, with a vector. In some embodiments, the vector is apseudotyped, self-inactivating lentiviral vector that carries exogenouspolynucleotide. In some embodiments, the vector is a self-inactivatinglentiviral vector pseudotyped with a vesicular stomatitis VSV-Genvelope, and which carries the exogenous polynucleotide. In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction. In some embodiments,exogenous polynucleotide is inserted into at least one allele of thecell using a lentivirus based viral vector. In some embodiments, theexogenous polynucleotide is inserted into a safe harbor or target locusof at least one allele of the cell.

An “exogenous” molecule is a molecule, construct, factor and the likethat is not normally present in a cell, but can be introduced into acell by one or more genetic, biochemical or other methods. “Normalpresence in the cell” is determined with respect to the particulardevelopmental stage and environmental conditions of the cell. Thus, forexample, a molecule that is present only during embryonic development ofneurons is an exogenous molecule with respect to an adult neuron cell.An exogenous molecule can comprise, for example, a functioning versionof a malfunctioning endogenous molecule or a malfunctioning version of anormally-functioning endogenous molecule.

An exogenous molecule or factor can be, among other things, a smallmolecule, such as is generated by a combinatorial chemistry process, ora macromolecule such as a protein, nucleic acid, carbohydrate, lipid,glycoprotein, lipoprotein, polysaccharide, any modified derivative ofthe above molecules, or any complex comprising one or more of the abovemolecules. Nucleic acids include DNA and RNA, can be single- ordouble-stranded; can be linear, branched or circular; and can be of anylength. Nucleic acids include those capable of forming duplexes, as wellas triplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule or construct can be the same type of molecule asan endogenous molecule, e.g., an exogenous protein or nucleic acid. Insuch instances, the exogenous molecule is introduced into the cell atgreater concentrations than that of the endogenous molecule in the cell.In some instances, an exogenous nucleic acid can comprise an infectingviral genome, a plasmid or episome introduced into a cell, or achromosome that is not normally present in the cell. Methods for theintroduction of exogenous molecules into cells are known to those ofskill in the art and include, but are not limited to, lipid-mediatedtransfer (i.e., liposomes, including neutral and cationic lipids),electroporation, direct injection, cell fusion, particle bombardment,calcium phosphate co-precipitation, DEAE-dextran-mediated transfer andviral vector-mediated transfer.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product, as well as all DNA regions whichregulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and/or locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristoylation, and/or glycosylation.

The term “genetic modification” and its grammatical equivalents as usedherein can refer to one or more alterations of a nucleic acid, e.g., thenucleic acid within an organism's genome. For example, geneticmodification can refer to alterations, additions, and/or deletion ofgenes or portions of genes or other nucleic acid sequences. Agenetically modified cell can also refer to a cell with an added,deleted and/or altered gene or portion of a gene. A genetically modifiedcell can also refer to a cell with an added nucleic acid sequence thatis not a gene or gene portion. Genetic modifications include, forexample, both transient knock-in or knock-down mechanisms, andmechanisms that result in permanent knock-in, knock-down, or knock-outof target genes or portions of genes or nucleic acid sequences Geneticmodifications include, for example, both transient knock-in andmechanisms that result in permanent knock-in of nucleic acids sequencesGenetic modifications also include, for example, reduced or increasedtranscription, reduced or increased mRNA stability, reduced or increasedtranslation, and reduced or increased protein stability.

As used herein, the terms “grafting”, “administering,” “introducing”,“implanting” and “transplanting” as well as grammatical variationsthereof are used interchangeably in the context of the placement ofcells (e.g., cells described herein) into a subject, by a method orroute which results in localization or at least partial localization ofthe introduced cells at a desired site or systemic introduction (e.g.,into circulation). The cells can be implanted directly to the desiredsite, or alternatively be administered by any appropriate route whichresults in delivery to a desired location in the subject where at leasta portion of the implanted cells or components of the cells remainviable. The period of viability of the cells after administration to asubject can be as short as a few hours, e.g. twenty-four hours, to a fewdays, to as long as several years. In some embodiments, the cells canalso be administered (e.g., injected) a location other than the desiredsite, such as in the brain or subcutaneously, for example, in a capsuleto maintain the implanted cells at the implant location and avoidmigration of the implanted cells.

By “HLA” or “human leukocyte antigen” complex is a gene complex encodingthe MHC proteins in humans. These cell-surface proteins that make up theHLA complex are responsible for the regulation of the immune response toantigens. In humans, there are two MHCs, class I and class II, “HLA-I”and “HLA-II”. HLA-I includes three proteins, HLA-A, HLA-B and HLA-C,which present peptides from the inside of the cell, and antigenspresented by the HLA-I complex attract killer T-cells (also known asCD8+ T-cells or cytotoxic T cells). The HLA-I proteins are associatedwith 3-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP,HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outsidethe cell to T lymphocytes. This stimulates CD4+ cells (also known asT-helper cells). It should be understood that the use of either “MHC” or“HLA” is not meant to be limiting, as it depends on whether the genesare from humans (HLA) or murine (MHC). Thus, as it relates to mammaliancells, these terms may be used interchangeably herein.

As used herein to characterize a cell, the term “hypoimmunogenic”generally means that such cell is less prone to innate or adaptiveimmune rejection by a subject into which such cells are transplanted,e.g., the cell is less prone to allorejection by a subject into whichsuch cells are transplanted. For example, relative to a cell of the samecell type that does not comprise the modifications, such ahypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to innate oradaptive immune rejection by a subject into which such cells aretransplanted. In some embodiments, genome editing technologies are usedto modulate the expression of MHC I and MHC II genes, and thus,contribute to generation of a hypoimmunogenic cell. In some embodiments,a hypoimmunogenic cell evades immune rejection in an MHC-mismatchedallogeneic recipient. In some instance, differentiated cells producedfrom the hypoimmunogenic stem cells outlined herein evade immunerejection when administered (e.g., transplanted or grafted) to anMHC-mismatched allogeneic recipient. In some embodiments, ahypoimmunogenic cell is protected from T cell-mediated adaptive immunerejection and/or innate immune cell rejection. Detailed descriptions ofhypoimmunogenic cells, methods of producing thereof, and methods ofusing thereof are found in WO2016183041 filed May 9, 2015; WO2018132783filed Jan. 14, 2018; WO2018176390 filed Mar. 20, 2018; WO2020018615filed Jul. 17, 2019; WO2020018620 filed Jul. 17, 2019; PCT/US2020/44635filed Jul. 31, 2020; U.S. 62/881,840 filed Aug. 1, 2019; U.S. 62/891,180filed Aug. 23, 2019; U.S. 63/016,190, filed Apr. 27, 2020; and U.S.63/052,360 filed Jul. 15, 2020, the disclosures including the examples,sequence listings and figures are incorporated herein by reference intheir entirety.

Hypoimmunogenicity of a cell can be determined by evaluating theimmunogenicity of the cell such as the cell's ability to elicit adaptiveand innate immune responses or to avoid eliciting such adaptive andinnate immune responses. Such immune response can be measured usingassays recognized by those skilled in the art. In some embodiments, animmune response assay measures the effect of a hypoimmunogenic cell on Tcell proliferation, T cell activation, T cell killing, donor specificantibody generation, NK cell proliferation, NK cell activation, andmacrophage activity. In some cases, hypoimmunogenic cells andderivatives thereof undergo decreased killing by T cells and/or NK cellsupon administration to a subject. In some instances, the cells andderivatives thereof show decreased macrophage engulfment compared to anunmodified or wild-type cell. In some embodiments, a hypoimmunogeniccell elicits a reduced or diminished immune response in a recipientsubject compared to a corresponding unmodified wild-type cell. In someembodiments, a hypoimmunogenic cell is nonimmunogenic or fails to elicitan immune response in a recipient subject.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refers to two or more sequences orsubsequences that have a specified percentage of nucleotides or aminoacid residues that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the sequence comparisonalgorithms described below (e.g., BLASTP and BLASTN or other algorithmsavailable to persons of skill) or by visual inspection. Depending on theapplication, the percent “identity” can exist over a region of thesequence being compared, e.g., over a functional domain, or,alternatively, exist over the full length of the two sequences to becompared. For sequence comparison, typically one sequence acts as areference sequence to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information.

“Immune signaling factor” as used herein refers to, in some cases, amolecule, protein, peptide and the like that activates immune signalingpathways.

“Immunosuppressive factor” or “immune regulatory factor” or “tolerogenicfactor” as used herein include hypoimmunity factors, complementinhibitors, and other factors that modulate or affect the ability of acell to be recognized by the immune system of a host or recipientsubject upon administration, transplantation, or engraftment. These maybe in combination with additional genetic modifications.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level. In some embodiments, the reference level, also referredto as the basal level, is 0.

In some embodiments, the alteration is an indel. As used herein, “indel”refers to a mutation resulting from an insertion, deletion, or acombination thereof. As will be appreciated by those skilled in the art,an indel in a coding region of a genomic sequence will result in aframeshift mutation, unless the length of the indel is a multiple ofthree. In some embodiments, the alteration is a point mutation. As usedherein, “point mutation” refers to a substitution that replaces one ofthe nucleotides. A gene editing (e.g., CRISPR/Cas) system of the presentdisclosure can be used to induce an indel of any length or a pointmutation in a target polynucleotide sequence.

As used herein, “knock down” refers to a reduction in expression of thetarget mRNA or the corresponding target protein. Knock down is commonlyreported relative to levels present following administration orexpression of a noncontrol molecule that does not mediate reduction inexpression levels of RNA (e.g., a non-targeting control shRNA, siRNA, ormiRNA). In some embodiments, knock down of a target gene is achieved byway of conditional or inducible shRNAs, conditional or inducible siRNAs,conditional or inducible miRNAs, or conditional or inducible CRISPRinterference (CRISPRi). In some embodiments, knock down of a target geneis achieved by way of a protein-based method, such as a conditional orinducible degron method. In some embodiments, knock down of a targetgene is achieved by genetic modification, including shRNAs, siRNAs,miRNAs, or use of gene editing systems (e.g., CRISPR/Cas).

Knock down is commonly assessed by measuring the mRNA levels usingquantitative polymerase chain reaction (qPCR) amplification or bymeasuring protein levels by western blot or enzyme-linked immunosorbentassay (ELISA). Analyzing the protein level provides an assessment ofboth mRNA cleavage as well as translation inhibition. Further techniquesfor measuring knock down include RNA solution hybridization, nucleaseprotection, northern hybridization, gene expression monitoring with amicroarray, antibody binding, radioimmunoassay, and fluorescenceactivated cell analysis. Those skilled in the art will readilyappreciate how to use the gene editing systems (e.g., CRISPR/Cas) of thepresent disclosure to knock out a target polynucleotide sequence or aportion thereof based upon the details described herein.

By “knock in” or “knock-in” herein is meant a genetic modificationresulting from the insertion of a DNA sequence into a chromosomal locusin a host cell. This causes initiation of or increased levels ofexpression of the knocked in gene, portion of gene, or nucleic acidsequence inserted product, e.g., an increase in RNA transcript levelsand/or encoded protein levels. As will be appreciated by those in theart, this can be accomplished in several ways, including inserting oradding one or more additional copies of the gene or portion thereof tothe host cell or altering a regulatory component of the endogenous geneincreasing expression of the protein is made or inserting a specificnucleic acid sequence whose expression is desired. This may beaccomplished by modifying a promoter, adding a different promoter,adding an enhancer, adding other regulatory elements, or modifying othergene expression sequences.

As used herein, “knock out” or “knock-out” includes deleting all or aportion of a target polynucleotide sequence in a way that interfereswith the translation or function of the target polynucleotide sequence.For example, a knock out can be achieved by altering a targetpolynucleotide sequence by inducing an insertion or a deletion (“indel”)in the target polynucleotide sequence, including in a functional domainof the target polynucleotide sequence (e.g., a DNA binding domain).Those skilled in the art will readily appreciate how to use the geneediting systems (e.g., CRISPR/Cas) of the present disclosure to knockout a target polynucleotide sequence or a portion thereof based upon thedetails described herein.

In some embodiments, a genetic modification or alteration results in aknock out or knock down of the target polynucleotide sequence or aportion thereof. Knocking out a target polynucleotide sequence or aportion thereof using a gene editing system (e.g., CRISPR/Cas) of thepresent disclosure can be useful for a variety of applications. Forexample, knocking out a target polynucleotide sequence in a cell can beperformed in vitro for research purposes. For ex vivo purposes, knockingout a target polynucleotide sequence in a cell can be useful fortreating or preventing a disorder associated with expression of thetarget polynucleotide sequence (e.g., by knocking out a mutant allele ina cell ex vivo and introducing those cells comprising the knocked outmutant allele into a subject) or for changing the genotype or phenotypeof a cell.

“Modulation” of gene expression refers to a change in the expressionlevel of a gene. Modulation of expression can include, but is notlimited to, gene activation and gene repression. Modulation may also becomplete, i.e., wherein gene expression is totally inactivated or isactivated to wild-type levels or beyond; or it may be partial, whereingene expression is partially reduced, or partially activated to somefraction of wild-type levels.

In additional or alternative aspects, the present disclosurecontemplates altering target polynucleotide sequences in any mannerwhich is available to the skilled artisan, e.g., utilizing a nucleasesystem such as a TAL effector nuclease (TALEN) or zinc finger nuclease(ZFN) system. It should be understood that although examples of methodsutilizing CRISPR/Cas (e.g., Cas9 and Cas12a) and TALEN are described indetail herein, the disclosure is not limited to the use of thesemethods/systems. Other methods of targeting to reduce or ablateexpression in target cells known to the skilled artisan can be utilizedherein. The methods provided herein can be used to alter a targetpolynucleotide sequence in a cell. The present disclosure contemplatesaltering target polynucleotide sequences in a cell for any purpose. Insome embodiments, the target polynucleotide sequence in a cell isaltered to produce a mutant cell. As used herein, a “mutant cell” refersto a cell with a resulting genotype that differs from its originalgenotype. In some instances, a “mutant cell” exhibits a mutantphenotype, for example when a normally functioning gene is altered usingthe gene editing systems (e.g., CRISPR/Cas) systems of the presentdisclosure. In other instances, a “mutant cell” exhibits a wild-typephenotype, for example when a gene editing system (e.g., CRISPR/Cas)system of the present disclosure is used to correct a mutant genotype.In some embodiments, the target polynucleotide sequence in a cell isaltered to correct or repair a genetic mutation (e.g., to restore anormal phenotype to the cell). In some embodiments, the targetpolynucleotide sequence in a cell is altered to induce a geneticmutation (e.g., to disrupt the function of a gene or genomic element).

The term “native cell” as used herein refers to a cell that is nototherwise modified (e.g., engineered). In some embodiments, a nativecell is a naturally occurring wild-type or a control cell.

The term “operatively linked” or “operably linked” are usedinterchangeably with reference to a juxtaposition of two or morecomponents (such as sequence elements), in which the components arearranged such that both components function normally and allow thepossibility that at least one of the components can mediate a functionthat is exerted upon at least one of the other components. By way ofillustration, a transcriptional regulatory sequence, such as a promoter,is operatively linked to a coding sequence if the transcriptionalregulatory sequence controls the level of transcription of the codingsequence in response to the presence or absence of one or moretranscriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

“Pluripotent stem cells” as used herein have the potential todifferentiate into any of the three germ layers: endoderm (e.g., thestomach linking, gastrointestinal tract, lungs, etc.), mesoderm (e.g.,muscle, bone, blood, urogenital tissue, etc.) or ectoderm (e.g.,epidermal tissues and nervous system tissues). The term “pluripotentstem cells,” as used herein, also encompasses “induced pluripotent stemcells”, or “iPSCs”, or a type of pluripotent stem cell derived from anon-pluripotent cell. In some embodiments, a pluripotent stem cell isproduced or generated from a cell that is not a pluripotent cell. Inother words, pluripotent stem cells can be direct or indirect progeny ofa non-pluripotent cell. Examples of parent cells include somatic cellsthat have been reprogrammed to induce a pluripotent, undifferentiatedphenotype by various means. Such “iPS” or “iPSC” cells can be created byinducing the expression of certain regulatory genes or by the exogenousapplication of certain proteins. Methods for the induction of iPS cellsare known in the art and are further described below. (See, e.g., Zhouet al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., NatureBiotechnol. 26 (7): 795 (2008); Woltjen et al., Nature 458 (7239):766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009); eachof which is incorporated by reference herein in their entirety.) Thegeneration of induced pluripotent stem cells (iPSCs) is outlined below.As used herein, “hiPSCs” are human induced pluripotent stem cells. Insome embodiments, “pluripotent stem cells,” as used herein, alsoencompasses mesenchymal stem cells (MSCs), and/or embryonic stem cells(ESCs).

As used herein, “promoter,” “promoter sequence,” or “promoter region”refers to a DNA regulatory region/sequence capable of binding RNApolymerase and involved in initiating transcription of a downstreamcoding or non-coding sequence. In some examples, the promoter sequenceincludes the transcription initiation site and extends upstream toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. In someembodiments, the promoter sequence includes a transcription initiationsite, as well as protein binding domains responsible for the binding ofRNA polymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

In some embodiments, the engineered and hypoimmunogenic cells describedare propagated from a primary T cell or a progeny thereof. As usedherein, the term “propagated from a primary T cell or a progeny thereof”encompasses the initial primary T cell that is isolated from the donorsubject and any subsequent progeny thereof. As used herein, the term“progeny” encompasses, e.g., a first-generation progeny, i.e., theprogeny is directly derived from, obtained from, obtainable from orderivable from the initial primary T cell by, e.g., traditionalpropagation methods. The term “progeny” also encompasses furthergenerations such as second, third, fourth, fifth, sixth, seventh, ormore generations, i.e., generations of cells which are derived from,obtained from, obtainable from or derivable from the former generationby, e.g., traditional propagation methods. The term “progeny” alsoencompasses modified cells that result from the modification oralteration of the initial primary T cell or a progeny thereof.

The term “recipient patient” refers to an animal, for example, a humanto whom treatment, including prophylactic treatment, with the cells asdescribed herein, is provided. For treatment of those infections,conditions or disease states, which are specific for a specific animalsuch as a human patient, the term patient refers to that specificanimal. The term “recipient patient” also encompasses any vertebrateincluding but not limited to mammals, reptiles, amphibians and fish.However, advantageously, the recipient patient is a mammal such as ahuman, or other mammals such as a domesticated mammal, e.g., dog, cat,horse, and the like, or production mammal, e.g., cow, sheep, pig, andthe like.

As used herein, the terms “regulatory sequences,” “regulatory elements,”and “control elements” are interchangeable and refer to polynucleotidesequences that are upstream (5′ non-coding sequences), within, ordownstream (3′ non-translated sequences) of a polynucleotide target tobe expressed. Regulatory sequences influence, for example but are notlimited to, the timing of transcription, amount or level oftranscription, RNA processing or stability, and/or translation of therelated structural nucleotide sequence. Regulatory sequences may includeactivator binding sequences, enhancers, introns, polyadenylationrecognition sequences, promoters, repressor binding sequences, stem-loopstructures, translational initiation sequences, translation leadersequences, transcription termination sequences, translation terminationsequences, primer binding sites, and the like. It is recognized thatsince in most cases the exact boundaries of regulatory sequences havenot been completely defined, nucleotide sequences of different lengthsmay have identical regulatory or promoter activity.

“Safe harbor locus” as used herein refers to a gene locus that allowsexpression of a transgene or an exogenous gene in a manner that enablesthe newly inserted genetic elements to function predictably and thatalso may not cause alterations of the host genome in a manner that posesa risk to the host cell. Exemplary “safe harbor” loci include, but arenot limited to, a CCR5 gene, a PPP1R12C (also known as AAVS1) gene, aCLYBL gene, and/or a Rosa gene (e.g., ROSA26).

“Target locus” as used herein refers to a gene locus that allowsexpression of a transgene or an exogenous gene. Exemplary “target loci”include, but are not limited to, a CXCR4 gene, an albumin gene, a SHS231locus, an F3 gene (also known as CD142), a MICA gene, a MICB gene, aLRP1 gene (also known as CD91), a HMGB1 gene, an ABO gene, a RHD gene, aFUT1 gene, and/or a KDM5D gene (also known as HY). The exogenouspolynucleotide encoding the exogenous gene can be inserted in the CDSregion for B2M, CIITA, TRAC, TRBC, CCR5, F3 (i.e., CD142), MICA, MICB,LRP1, HMGB1, ABO, RHD, FUT1, KDM5D (i.e., HY), PDGFRa, OLIG2, and/orGFAP. The exogenous polynucleotide encoding the exogenous gene can beinserted in introns 1 or 2 for PPP1R12C (i.e., AAVS1) or CCR5. Theexogenous polynucleotide encoding the exogenous gene can be inserted inexons 1 or 2 or 3 for CCR5. The exogenous polynucleotide encoding theexogenous gene can be inserted in intron 2 for CLYBL. The exogenouspolynucleotide encoding the exogenous gene can be inserted in a 500 bpwindow in Ch-4:58,976,613 (i.e., SHS231). The exogenous polynucleotideencoding the exogenous gene can be insert in any suitable region of theaforementioned safe harbor or target loci that allows for expression ofthe exogenous gene, including, for example, an intron, an exon or acoding sequence region in a safe harbor or target locus.

As used herein, a “target” can refer to a gene, a portion of a gene, aportion of the genome, or a protein that is subject to regulatablereduced expression by the methods described herein.

As used herein, “therapeutically effective amount” refers to an amountsufficient to provide a therapeutic benefit in the treatment and/ormanagement of a disease, disorder, or condition. In some embodiments, atherapeutically effective amount is an amount sufficient to ameliorate,palliate, stabilize, reverse, slow, attenuate or delay the progressionof a disease, disorder, or condition, or of a symptom or side effect ofthe disease, disorder, or condition. In some embodiments, thetherapeutically effective amount is also a clinically effective amount.In other embodiments, the therapeutically effective amount is not aclinically effective amount.

As used herein, the term “treating” and “treatment” includesadministering to a subject a therapeutically or clinically effectiveamount of cells described herein so that the subject has a reduction inat least one symptom of the disease or an improvement in the disease,for example, beneficial or desired therapeutic or clinical results. Forpurposes of this technology, beneficial or desired therapeutic orclinical results include, but are not limited to, alleviation of one ormore symptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Treating canrefer to prolonging survival as compared to expected survival if notreceiving treatment. Thus, one of skill in the art realizes that atreatment may improve the disease condition, but may not be a completecure for the disease. In some embodiments, one or more symptoms of acondition, disease or disorder are alleviated by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, or at least 50% upontreatment of the condition, disease or disorder.

For purposes of this technology, beneficial or desired therapeutic orclinical results of disease treatment include, but are not limited to,alleviation of one or more symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable.

A “vector” or “construct” is capable of transferring gene sequences totarget cells. Typically, “vector construct,” “expression vector,” and“gene transfer vector,” mean any nucleic acid construct capable ofdirecting the expression of a gene of interest and which can transfergene sequences to target cells. Thus, the term includes cloning, andexpression vehicles, as well as integrating vectors. Methods for theintroduction of vectors or constructs into cells are known to those ofskill in the art and include, but are not limited to, lipid-mediatedtransfer (i.e., liposomes, including neutral and cationic lipids),electroporation, direct injection, cell fusion, particle bombardment,calcium phosphate co-precipitation, DEAE-dextran-mediated transferand/or viral vector-mediated transfer.

In some embodiments, the cells are engineered to have reduced orincreased expression of one or more targets relative to an unaltered orunmodified wild-type cell. In some embodiments, the cells are engineeredto have constitutive reduced or increased expression of one or moretargets relative to an unaltered or unmodified wild-type cell. In someembodiments, the cells are engineered to have regulatable reduced orincreased expression of one or more targets relative to an unaltered orunmodified wild-type cell. In some embodiments, the cells compriseincreased expression of CD47 relative to a wild-type cell or a controlcell of the same cell type. By “wild-type” or “wt” or “control” in thecontext of a cell means any cell found in nature. Examples of wild typeor control cells include primary cells and T cells found in nature.However, by way of example, in the context of an engineered cell, asused herein, “wild-type” or “control” can also mean an engineered cellthat may contain nucleic acid changes resulting in reduced expression ofMHC I and/or II and/or T-cell receptors, but did not undergo the geneediting procedures to result in overexpression of CD47 proteins. Forexample, as used herein, “wild-type” or “control” means an engineeredcell that comprises reduced or knocked out expression of B2M, CIITA,and/or TRAC. Also as used herein, “wild-type” or “control” means anengineered cell that comprises reduced or knocked out expression of B2M,CIITA, TRAC, and/or TRBC. As used herein, “wild-type” or “control” alsomeans an engineered cell that may contain nucleic acid changes resultingin overexpression of CD47 proteins, but did not undergo the gene editingprocedures to result in reduced expression of MHC I and/or II and/orT-cell receptors. In the context of an iPSC or a progeny thereof,“wild-type” or “control” also means an iPSC or progeny thereof that maycontain nucleic acid changes resulting in pluripotency but did notundergo the gene editing procedures of the present disclosure to achievereduced expression of MHC I and/or II and/or T-cell receptors, and/oroverexpression of CD47 proteins. For example, as used herein,“wild-type” or “control” means an iPSC or progeny thereof that comprisesreduced or knocked out expression of B2M, CIITA, and/or TRAC. Also asused herein, “wild-type” or “control” means an iPSC or progeny thereofthat comprises reduced or knocked out expression of B2M, CIITA, TRAC,and/or TRBC. In the context of a primary T cell or a progeny thereof,“wild-type” or “control” also means a primary T cell or progeny thereofthat may contain nucleic acid changes resulting in reduced expression ofMHC I and/or II and/or T-cell receptors, but did not undergo the geneediting procedures to result in overexpression of CD47 proteins. Forexample, as used herein, “wild-type” or “control” means a primary T cellor progeny thereof that comprises reduced or knocked out expression ofB2M, CIITA, and/or TRAC. Also as used herein, “wild-type” or “control”means a primary T cell or progeny thereof that comprises reduced orknocked out expression of B2M, CIITA, TRAC, and/or TRBC. Also in thecontext of a primary T cell or a progeny thereof, “wild-type” or“control” also means a primary T cell or progeny thereof that maycontain nucleic acid changes resulting in overexpression of CD47proteins, but did not undergo the gene editing procedures to result inreduced expression of MHC I and/or II and/or T-cell receptors. In someembodiments, the cells are engineered to have regulatable reduced orincreased expression of one or more targets relative to a cell of thesame cell type that does not comprise the modifications. In someembodiments, the wild-type cell or the control cell is a startingmaterial. In some embodiments, the starting material is a primary cellcollected from a donor. In some embodiments, the starting material is aprimary blood cell collected from a donor, e.g., via a leukopak. Forexample, unmodified T cells obtained from a donor is a starting materialthat are considered wild-type or control cells as contemplated herein.In another example, an iPSC cell line starting material is a startingmaterial that is considered a wild-type or control cell as contemplatedherein. In some embodiments, the starting material is otherwise modifiedor engineered to have altered expression of one or more genes togenerate the engineered cell.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodimentsdescribed and illustrated herein has discrete components and featuresreadily separated from or combined with the features of any of the otherseveral embodiments without departing from the scope or spirit of thepresent disclosure. Any recited method may be carried out in the orderof events recited or in any other order that is logically possible.Although any methods and materials similar or equivalent to thosedescribed herein may also be used in the practice or testing of thepresent disclosure, representative illustrative methods and materialsare now described.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this technology belongs. Where a range of values isprovided, it is understood that each intervening value, to the tenth ofthe unit of the lower limit unless the context clearly dictatesotherwise, between the upper and lower limit of that range and any otherstated or intervening value in that stated range, is encompassed withinthe present disclosure. The upper and lower limits of these smallerranges may independently be included in the smaller ranges and are alsoencompassed within the present disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the present disclosure. Certain ranges arepresented herein with numerical values being preceded by the term“about.” The term “about” is used herein to provide literal support forthe exact number that it precedes, as well as a number that is near toor approximately the number that the term precedes. In determiningwhether a number is near to or approximately a specifically recitednumber, the near or approximating unrecited number may be a number,which, in the context presented, provides the substantial equivalent ofthe specifically recited number. The term about is used herein to meanplus or minus ten percent (10%) of a value. For example, “about 100”refers to any number between 90 and 110.

All publications, patents, and patent applications cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application werespecifically and individually indicated to be incorporated by reference.Furthermore, each cited publication, patent, or patent application isincorporated herein by reference to disclose and describe the subjectmatter in connection with which the publications are cited. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the technology describedherein is not entitled to antedate such publication by virtue of priortechnology. Further, the dates of publication provided might bedifferent from the actual publication dates, which may need to beindependently confirmed.

Before the technology is further described, it is to be understood thatthis technology is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present disclosure will be limited only by the appended claims. Itshould also be understood that the headers used herein are not limitingand are merely intended to orient the reader, but the subject mattergenerally applies to the technology disclosed herein.

III. Detailed Description of the Embodiments

A. Hypoimmunogenic Cells

In some embodiments, the present disclosure is directed to pluripotentstem cells (e.g., pluripotent stem cells and induced pluripotent stemcells (iPSCs)), differentiated cells derived from such pluripotent stemcells (such as, but not limited to, T cells and NK cells), and primarycells (such as, but not limited to, primary T cells and primary NKcells). In some embodiments, the pluripotent stem cells, differentiatedcells derived therefrom, such as T cells and NK cells, and primary cellssuch as primary T cells and primary NK cells, are engineered for reducedexpression or lack of expression of MHC class I and/or MHC class IIhuman leukocyte antigens, and in some instances, for reduced expressionor lack of expression of a T-cell receptor (TCR) complex. In someembodiments, the hypoimmune (HIP) T cells and primary T cellsoverexpress CD47 and a chimeric antigen receptor (CAR) in addition toreduced expression or lack of expression of MHC class I and/or MHC classII human leukocyte antigens, and have reduced expression or lackexpression of a T-cell receptor (TCR) complex. In some embodiments, theCAR comprises an antigen binding domain that binds to any one selectedfrom the group consisting of CD19, CD22, CD38, CD123, CD138, and BCMA.In some embodiments, the CAR is a CD19-specific CAR. In someembodiments, the CAR is a CD22-specific CAR. In some instances, the CARis a CD38-specific CAR. In some embodiments, the CAR is a CD123-specificCAR. In some embodiments, the CAR is a CD138-specific CAR. In someinstances, the CAR is a BCMA-specific CAR. In some embodiments, the CARis a bispecific CAR. In some embodiments, the bispecific CAR is aCD19/CD22-bispecific CAR. In some embodiments, the bispecific CAR is aBCMA/CD38-bispecific CAR. In some embodiments, the cells describedexpress a CD19-specific CAR and a different CAR, such as, but notlimited to a CD22-specific CAR, a CD38-specific CAR, a CD123-specificCAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments,the cells described express a CD22-specific CAR and a different CAR,such as, but not limited to a CD19-specific CAR, a CD38-specific CAR, aCD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. Insome embodiments, the cells described express a CD38-specific CAR and adifferent CAR, such as, but not limited to a CD22-specific CAR, aCD18-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and aBCMA-specific CAR. In some embodiments, the cells described express aCD123-specific CAR and a different CAR, such as, but not limited to aCD22-specific CAR, a CD38-specific CAR, a CD19-specific CAR, aCD138-specific CAR, and a BCMA-specific CAR. In some embodiments, thecells described express a CD138-specific CAR and a different CAR, suchas, but not limited to a CD22-specific CAR, a CD38-specific CAR, aCD123-specific CAR, a CD19-specific CAR, and a BCMA-specific CAR. Insome embodiments, the cells described express a BCMA-specific CAR and adifferent CAR, such as, but not limited to a CD22-specific CAR, aCD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and aCD19-specific CAR. In some embodiments, the cells are modified orengineered as compared to a wild-type or control cell, including anunaltered or unmodified wild-type cell or control cell. In someembodiments, the wild-type cell or the control cell is a startingmaterial. In some embodiments, the starting material is a primary cellcollected from a donor. In some embodiments, the starting material is aprimary blood cell collected from a donor, e.g., via a leukopak. In someembodiments, the starting material is otherwise modified or engineeredto have altered expression of one or more genes to generate theengineered cell.

In some embodiments, engineered and/or hypoimmune (HIP) T cells andprimary T cells overexpress CD47 and a chimeric antigen receptor (CAR),and include a genomic modification of the B2M gene. In some embodiments,engineered and/or hypoimmune (HIP) T cells and primary T cellsoverexpress CD47 and include a genomic modification of the CIITA gene.In some embodiments, engineered and/or hypoimmune (HIP) T cells andprimary T cells overexpress CD47 and a CAR, and include a genomicmodification of the TRAC gene. In some embodiments, engineered and/orhypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR,and include a genomic modification of the TRB gene. In some embodiments,engineered and/or hypoimmune (HIP) T cells and primary T cellsoverexpress CD47 and a CAR, and include one or more genomicmodifications selected from the group consisting of the B2M, CIITA,TRAC, and TRB genes. In some embodiments, engineered and/or hypoimmune(HIP) T cells and primary T cells overexpress CD47 and a CAR, andinclude genomic modifications of the B2M, CIITA, TRAC, and TRB genes. Insome embodiments, the cells are B2M^(−/−), CIITA^(−/−), TRAC^(−/−),CD47tg cells that also express CARs. In some embodiments, engineeredand/or hypoimmune (HIP) T cells are produced by differentiating inducedpluripotent stem cells such as engineered and/or hypoimmunogenic inducedpluripotent stem cells. In some embodiments, the cells are modified orengineered as compared to a wild-type or control cell, including anunaltered or unmodified wild-type cell or control cell. In someembodiments, the wild-type cell or the control cell is a startingmaterial. In some embodiments, the starting material is a primary cellcollected from a donor. In some embodiments, the starting material is aprimary blood cell collected from a donor, e.g., via a leukopak. In someembodiments, the starting material is otherwise modified or engineeredto have altered expression of one or more genes to generate theengineered cell.

In some embodiments, the engineered and/or hypoimmune (HIP) T cells andprimary T cells are B2M^(−/−), CIITA^(−/−), TRB^(−/−), CD47tg cells thatalso express CARs. In some embodiments, the cells are B2M^(−/−),CIITA^(−/−), TRAC^(−/−), TRB^(−/−), CD47tg cells that also express CARs.In certain embodiments, the cells are B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) CD47tg cells that also expressCARs. In certain embodiments, the cells are B2M^(indel/indel),CIITA^(indel/indel) TRB^(indel/indel) CD47tg cells that also expressCARs. In certain embodiments, the cells are B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), TRB^(indel/indel), CD47tg cellsthat also express CARs. In some embodiments, the engineered or modifiedcells described are pluripotent stem cells, induced pluripotent stemcells, NK cells differentiated from such pluripotent stem cells andinduced pluripotent stem cells, T cells differentiated from suchpluripotent stem cells and induced pluripotent stem cells, or primary Tcells. Non-limiting examples of primary T cells include CD3+ T cells,CD4+ T cells, CD8+ T cells, naïve T cells, regulatory T (Treg) cells,non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells,T-follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTL), effectorT (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem)cells, effector memory T cells express CD45RA (TEMRA cells),tissue-resident memory (Trm) cells, virtual memory T cells, innatememory T cells, memory stem cell (Tsc), γδ T cells, and any othersubtype of T cells. In some embodiments, the primary T cells areselected from a group that includes cytotoxic T-cells, helper T-cells,memory T-cells, regulatory T-cells, tumor infiltrating lymphocytes, andcombinations thereof. Non-limiting examples of NK cells and primary NKcells include immature NK cells and mature NK cells. In someembodiments, the cells are modified or engineered as compared to awild-type or control cell, including an unaltered or unmodifiedwild-type cell or control cell. In some embodiments, the wild-type cellor the control cell is a starting material. In some embodiments, thestarting material is a primary cell collected from a donor. In someembodiments, the starting material is a primary blood cell collectedfrom a donor, e.g., via a leukopak. In some embodiments, the startingmaterial is otherwise modified or engineered to have altered expressionof one or more genes to generate the engineered cell.

In some embodiments, the primary T cells are from a pool of primary Tcells from one or more donor subjects that are different than therecipient subject (e.g., the patient administered the cells). Theprimary T cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,50, 100 or more donor subjects and pooled together. The primary T cellscan be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10, or more 20 ormore, 50 or more, or 100 or more donor subjects and pooled together. Insome embodiments, the primary T cells are harvested from one or aplurality of individuals, and in some instances, the primary T cells orthe pool of primary T cells are cultured in vitro. In some embodiments,the primary T cells or the pool of primary T cells are engineered toexogenously express CD47 and cultured in vitro.

In certain embodiments, the primary T cells or the pool of primary Tcells are engineered to express a chimeric antigen receptor (CAR). TheCAR can be any known to those skilled in the art. Useful CARs includethose that bind an antigen selected from a group that includes CD19,CD20, CD22, CD38, CD123, CD138, and BCMA. In some cases, the CAR is thesame or equivalent to those used in FDA-approved CAR-T cell therapiessuch as, but not limited to, those used in tisagenlecleucel andaxicabtagene ciloleucel, or others under investigation in clinicaltrials.

In some embodiments, the primary T cells or the pool of primary T cellsare engineered to exhibit reduced expression of an endogenous T cellreceptor compared to unmodified primary T cells. In certain embodiments,the primary T cells or the pool of primary T cells are engineered toexhibit reduced expression of CTLA-4, PD-1, or both CTLA-4 and PD-1, ascompared to unmodified primary T cells. Methods of genetically modifyinga cell including a T cell are described in detail, for example, inWO2020/018620 and WO2016/183041, the disclosures of which are hereinincorporated by reference in their entireties, including the tables,appendices, sequence listing and figures.

In some embodiments, the CAR-T cells comprise a CAR selected from agroup including: (a) a first generation CAR comprising an antigenbinding domain, a transmembrane domain, and a signaling domain; (b) asecond generation CAR comprising an antigen binding domain, atransmembrane domain, and at least two signaling domains; (c) a thirdgeneration CAR comprising an antigen binding domain, a transmembranedomain, and at least three signaling domains; and (d) a fourthgeneration CAR comprising an antigen binding domain, a transmembranedomain, three or four signaling domains, and a domain which uponsuccessful signaling of the CAR induces expression of a cytokine gene.

In some embodiments, the CAR-T cells comprise a CAR comprising anantigen binding domain, a transmembrane, and one or more signalingdomains. In some embodiments, the CAR also comprises a linker. In someembodiments, the CAR comprises a CD19 antigen binding domain. In someembodiments, the CAR comprises a CD28 or a CD8a transmembrane domain. Insome embodiments, the CAR comprises a CD8a signal peptide. In someembodiments, the CAR comprises a Whitlow linker GSTSGSGKPGSGEGSTKG (SEQID NO: 15). In some embodiments, the antigen binding domain of the CARis selected from a group including, but not limited to, (a) an antigenbinding domain targets an antigen characteristic of a neoplastic cell;(b) an antigen binding domain that targets an antigen characteristic ofa T cell; (c) an antigen binding domain targets an antigencharacteristic of an autoimmune or inflammatory disorder; (d) an antigenbinding domain that targets an antigen characteristic of senescentcells; (e) an antigen binding domain that targets an antigencharacteristic of an infectious disease; and (f) an antigen bindingdomain that binds to a cell surface antigen of a cell.

In some embodiments, the CAR further comprises one or more linkers. Theformat of an scFv is generally two variable domains linked by a flexiblepeptide sequence, or a “linker,” either in the orientation VH-linker-VLor VL-linker-VH. Any suitable linker known to those in the art in viewof the specification can be used in the CARs. Examples of suitablelinkers include, but are not limited to, a GS based linker sequence, anda Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO:15). In some embodiments,the linker is a GS or a gly-ser linker. Exemplary gly-ser polypeptidelinkers comprise the amino acid sequence Ser(Gly₄Ser)_(n), as well as(Gly₄Ser)_(n) and/or (Gly₄Ser₃)_(n). In some embodiments, n=1. In someembodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly₄Ser)₃. In someembodiments, n=4, i.e., Ser(Gly₄Ser)₄. In some embodiments, n=5. In someembodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. Insome embodiments, n=9. In some embodiments, n=10. Another exemplarygly-ser polypeptide linker comprises the amino acid sequenceSer(Gly₄Ser)_(n). In some embodiments, n=1. In some embodiments, n=2. Insome embodiments, n=3. In another embodiment, n=4. In some embodiments,n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptidelinker comprises (Gly₄Ser)_(n). In some embodiments, n=1. In someembodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. Insome embodiments, n=5. In some embodiments, n=6. Another exemplarygly-ser polypeptide linker comprises (Gly₃Ser)_(n). In some embodiments,n=1. In some embodiments, n=2. In some embodiments, n=3. In someembodiments, n=4. In another embodiment, n=5. In yet another embodiment,n=6. Another exemplary gly-ser polypeptide linker comprises(Gly₄Ser₃)_(n). In some embodiments, n=1. In some embodiments, n=2. Insome embodiments, n=3. In some embodiments, n=4. In some embodiments,n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptidelinker comprises (Gly₃Ser)_(n). In some embodiments, n=1. In someembodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. Inanother embodiment, n=5. In yet another embodiment, n=6.

In some embodiments, the antigen binding domain is selected from a groupthat includes an antibody, an antigen-binding portion or fragmentthereof, an scFv, and a Fab. In some embodiments, the antigen bindingdomain binds to CD19, CD20, CD22, CD38, CD123, CD138, or BCMA. In someembodiments, the antigen binding domain is an anti-CD19 scFv such as butnot limited to FMC63.

In some embodiments, the transmembrane domain comprises one selectedfrom a group that includes a transmembrane region of TCRα, TCRβ, TCRζ,CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD28, CD45,CD22, CD33, CD34, CD37, CD40, CD40L/CD154, CD45, CD64, CD80, CD86,OX40/CD134, 4-1BB/CD137, CD154, FcεRIγ, VEGFR2, FAS, FGFR2B, andfunctional variant thereof.

In some embodiments, the signaling domain(s) of the CAR comprises acostimulatory domain(s). For instance, a signaling domain can contain acostimulatory domain. Or, a signaling domain can contain one or morecostimulatory domains. In certain embodiments, the signaling domaincomprises a costimulatory domain. In other embodiments, the signalingdomains comprise costimulatory domains. In some cases, when the CARcomprises two or more costimulatory domains, two costimulatory domainsare not the same. In some embodiments, the costimulatory domainscomprise two costimulatory domains that are not the same. In someembodiments, the costimulatory domain enhances cytokine production,CAR-T cell proliferation, and/or CAR-T cell persistence during T cellactivation. In some embodiments, the costimulatory domains enhancecytokine production, CAR-T cell proliferation, and/or CAR-T cellpersistence during T cell activation.

As described herein, a fourth generation CAR can contain an antigenbinding domain, a transmembrane domain, three or four signaling domains,and a domain which upon successful signaling of the CAR inducesexpression of a cytokine gene. In some instances, the cytokine gene isan endogenous or exogenous cytokine gene of the hypoimmunogenic cells.In some cases, the cytokine gene encodes a pro-inflammatory cytokine. Insome embodiments, the pro-inflammatory cytokine is selected from a groupthat includes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and afunctional fragment thereof. In some embodiments, the domain which uponsuccessful signaling of the CAR induces expression of the cytokine genecomprises a transcription factor or functional domain or fragmentthereof.

In some embodiments, the CAR comprises a CD3 zeta (CD3ζ) domain or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof. In some embodiments, the CAR comprises (i) a CD3 zetadomain, or an immunoreceptor tyrosine-based activation motif (ITAM), orfunctional variant thereof, and (ii) a CD28 domain, or a 4-1BB domain,or functional variant thereof. In other embodiments, the CAR comprises(i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activationmotif (ITAM), or functional variant thereof; (ii) a CD28 domain orfunctional variant thereof, and (iii) a 4-1BB domain, or a CD134 domain,or functional variant thereof. In certain embodiments, the CAR comprises(i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activationmotif (ITAM), or functional variant thereof; (ii) a CD28 domain orfunctional variant thereof, (iii) a 4-1BB domain, or a CD134 domain, orfunctional variant thereof, and (iv) a cytokine or costimulatory ligandtransgene. In some embodiments, the CAR comprises a (i) an anti-CD19scFv; (ii) a CD8a hinge and transmembrane domain or functional variantthereof, (iii) a 4-1BB costimulatory domain or functional variantthereof; and (iv) a CD3ζ signaling domain or functional variant thereof.

Methods for introducing a CAR construct or producing a CAR-T cells arewell known to those skilled in the art. Detailed descriptions are found,for example, in Vormittag et al., Curr Opin Biotechnol, 2018, 53,162-181; and Eyquem et al., Nature, 2017, 543, 113-117.

In some embodiments, the cells derived from primary T cells comprisereduced expression of an endogenous T cell receptor, for example bydisruption of an endogenous T cell receptor gene (e.g., T cell receptoralpha constant region (TRAC) or T cell receptor beta constant region(TRB)). In some embodiments, an exogenous nucleic acid encoding apolypeptide as disclosed herein (e.g., a chimeric antigen receptor,CD47, or another tolerogenic factor disclosed herein) is inserted at thedisrupted T cell receptor gene. In some embodiments, an exogenousnucleic acid encoding a polypeptide is inserted at a TRAC or a TRB genelocus.

In some embodiments, the cells derived from primary T cells comprisereduced expression of cytotoxic T-lymphocyte-associated protein 4(CTLA4) and/or programmed cell death (PD1). Methods of reducing oreliminating expression of CTLA4, PD1 and both CTLA4 and PD1 can includeany recognized by those skilled in the art, such as but not limited to,genetic modification technologies that utilize rare-cuttingendonucleases and RNA silencing or RNA interference technologies.Non-limiting examples of a rare-cutting endonuclease include any Casprotein, TALEN, zinc finger nuclease, meganuclease, and/or homingendonuclease. In some embodiments, an exogenous nucleic acid encoding apolypeptide as disclosed herein (e.g., a chimeric antigen receptor,CD47, or another tolerogenic factor disclosed herein) is inserted at aCTLA4 and/or PD1 gene locus. In some embodiments, the cells are modifiedor engineered as compared to a wild-type or control cell, including anunaltered or unmodified wild-type cell or control cell. In someembodiments, the wild-type cell or the control cell is a startingmaterial. In some embodiments, the starting material is a primary cellcollected from a donor. In some embodiments, the starting material is aprimary blood cell collected from a donor, e.g., via a leukopak. In someembodiments, the starting material is otherwise modified or engineeredto have altered expression of one or more genes to generate theengineered cell. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction,for example, with a vector. In some embodiments, the vector is apseudotyped, self-inactivating lentiviral vector that carries theexogenous polynucleotide. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, a CD47 transgene is inserted into a pre-selectedlocus of the cell. In some embodiments, a CD47 transgene is insertedinto a random locus of the cell. In some embodiments, a transgeneencoding a CAR is inserted into a pre-selected locus of the cell. Insome embodiments, a transgene encoding a CAR is inserted into a randomlocus of the cell. In certain embodiments, a CD47 transgene and atransgene encoding a CAR are inserted into a pre-selected locus of thecell. In some embodiments, a transgene encoding a CAR is inserted into arandom or pre-selected locus of the cell, including a safe harbor locus,via viral vector transduction/integration. In some embodiments, a CD47transgene and a transgene encoding a CAR are inserted into a random orpre-selected locus of the cell, including a safe harbor locus, via viralvector transduction/integration. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope. In some embodiments, the transgene encoding aCAR is inserted into at least one allele of the cell using viraltransduction. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using a lentivirus basedviral vector. The random and/or pre-selected locus can be a safe harboror target locus. Non-limiting examples of a safe harbor locus include,but are not limited to, a CCR5 gene locus, a PPP1R12C (also known asAAVS1) gene locus, and a CLYBL gene locus, a Rosa gene locus (e.g.,ROSA26 gene locus). Non-limiting examples of a target locus include, butare not limited to, a CXCR4 gene locus, an albumin gene locus, a SHS231gene locus, an F3 gene locus (also known as CD142), a MICA gene locus, aMICB gene locus, a LRP1 gene locus (also known as a CD91 gene locus), aHMGB1 gene locus, an ABO gene locus, ad RHD gene locus, a FUT1 locus,and a KDM5D gene locus. The CD47 transgene can be inserted in Introns 1or 2 for PPP1R12C (i.e., AAVS1) or CCR5. The CD47 transgene can beinserted in Exons 1 or 2 or 3 for CCR5. The CD47 transgene can beinserted in intron 2 for CLYBL. The CD47 transgene can be inserted in a500 bp window in Ch-4:58,976,613 (i.e., SHS231). The CD47 transgene canbe insert in any suitable region of the aforementioned safe harbor ortarget loci that allows for expression of the exogenous polynucleotide,including, for example, an intron, an exon or a coding sequence regionin a safe harbor or target locus. In some embodiments, the pre-selectedlocus is selected from the group consisting of the B2M locus, the CIITAlocus, the TRAC locus, and the TRB locus. In some embodiments, thepre-selected locus is the B2M locus. In some embodiments, thepre-selected locus is the CIITA locus. In some embodiments, thepre-selected locus is the TRAC locus. In some embodiments, thepre-selected locus is the TRB locus. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, a CD47 transgene and a transgene encoding a CAR areinserted into the same locus. In some embodiments, a CD47 transgene anda transgene encoding a CAR are inserted into different loci. In manyinstances, a CD47 transgene is inserted into a safe harbor or targetlocus. In many instances, a transgene encoding a CAR is inserted into asafe harbor or target locus. In some instances, a CD47 transgene isinserted into a B2M locus. In some instances, a transgene encoding a CARis inserted into a B2M locus. In certain instances, a CD47 transgene isinserted into a CIITA locus. In certain instances, a transgene encodinga CAR is inserted into a CIITA locus. In particular instances, a CD47transgene is inserted into a TRAC locus. In particular instances, atransgene encoding a CAR is inserted into a TRAC locus. In many otherinstances, a CD47 transgene is inserted into a TRB locus. In many otherinstances, a transgene encoding a CAR is inserted into a TRB locus. Insome embodiments, a CD47 transgene and a transgene encoding a CAR areinserted into a safe harbor or target locus (e.g., a CCR5 gene locus, aCXCR4 gene locus, a PPP1R12C gene locus, an albumin gene locus, a SHS231gene locus, a CLYBL gene locus, a Rosa gene locus, an F3 (CD142) genelocus, a MICA gene locus, a MICB gene locus, a LRP1 (CD91) gene locus, aHMGB1 gene locus, an ABO gene locus, an RHD gene locus, a FUT1 locus,and a KDM5D gene locus.

In certain embodiments, a CD47 transgene and a transgene encoding a CARare inserted into a safe harbor or target locus. In certain embodiments,a CD47 transgene and a transgene encoding a CAR are controlled by asingle promoter and are inserted into a safe harbor or target locus. Incertain embodiments, a CD47 transgene and a transgene encoding a CAR arecontrolled by their own promoters and are inserted into a safe harbor ortarget locus. In certain embodiments, a CD47 transgene and a transgeneencoding a CAR are inserted into a TRAC locus. In certain embodiments, aCD47 transgene and a transgene encoding a CAR are controlled by a singlepromoter and are inserted into a TRAC locus. In certain embodiments, aCD47 transgene and a transgene encoding a CAR are controlled by theirown promoters and are inserted into a TRAC locus. In some embodiments, aCD47 transgene and a transgene encoding a CAR are inserted into a TRBlocus. In some embodiments, a CD47 transgene and a transgene encoding aCAR are controlled by a single promoter and are inserted into a TRBlocus. In some embodiments, a CD47 transgene and a transgene encoding aCAR are controlled by their own promoters and are inserted into a TRBlocus. In other embodiments, a CD47 transgene and a transgene encoding aCAR are inserted into a B2M locus. In other embodiments, a CD47transgene and a transgene encoding a CAR are controlled by a singlepromoter and are inserted into a B2M locus. In other embodiments, a CD47transgene and a transgene encoding a CAR are controlled by their ownpromoters and are inserted into a B2M locus. In various embodiments, aCD47 transgene and a transgene encoding a CAR are inserted into a CIITAlocus. In various embodiments, a CD47 transgene and a transgene encodinga CAR are controlled by a single promoter and are inserted into a CIITAlocus. In various embodiments, a CD47 transgene and a transgene encodinga CAR are controlled by their own promoters and are inserted into aCIITA locus. In some instances, the promoter controlling expression ofany transgene described is a constitutive promoter. In other instances,the promoter for any transgene described is an inducible promoter. Insome embodiments, the promoter is an EF1α promoter. In some embodiments,the promoter is CAG promoter. In some embodiments, a CD47 transgene anda transgene encoding a CAR are both controlled by a constitutivepromoter. In some embodiments, a CD47 transgene and a transgene encodinga CAR are both controlled by an inducible promoter. In some embodiments,a CD47 transgene is controlled by a constitutive promoter and atransgene encoding a CAR is controlled by an inducible promoter. In someembodiments, a CD47 transgene is controlled by an inducible promoter anda transgene encoding a CAR is controlled by a constitutive promoter. Invarious embodiments, a CD47 transgene is controlled by an EF1α promoterand a transgene encoding a CAR is controlled by an EF1α promoter. Insome embodiments, a CD47 transgene is controlled by a CAG promoter and atransgene encoding a CAR is controlled by a CAG promoter. In someembodiments, a CD47 transgene is controlled by a CAG promoter and atransgene encoding a CAR is controlled by an EF1α promoter. In someembodiments, a CD47 transgene is controlled by an EF1α promoter and atransgene encoding a CAR is controlled by a CAG promoter. In someembodiments, expression of both a CD47 transgene and a transgeneencoding a CAR is controlled by a single EF1α promoter. In someembodiments, expression of both a CD47 transgene and a transgeneencoding a CAR is controlled by a single CAG promoter.

In another embodiment, the present disclosure disclosed herein isdirected to pluripotent stem cells, (e.g., pluripotent stem cells andinduced pluripotent stem cells (iPSCs)), differentiated cells derivedfrom such pluripotent stem cells (e.g., hypoimmune (HIP) T cells), andprimary T cells that overexpress CD47 (such as exogenously express CD47proteins), have reduced expression or lack expression of MHC class Iand/or MHC class II human leukocyte antigens, and have reducedexpression or lack expression of a T-cell receptor (TCR) complex. Insome embodiments, the hypoimmune (HIP) T cells and primary T cellsoverexpress CD47 (such as exogenously express CD47 proteins), havereduced expression or lack expression of MHC class I and/or MHC class IIhuman leukocyte antigens, and have reduced expression or lack expressionof a T-cell receptor (TCR) complex.

In some embodiments, pluripotent stem cells, (e.g., pluripotent stemcells and induced pluripotent stem cells (iPSCs)), differentiated cellsderived from such pluripotent stem cells (e.g., hypoimmune (HIP) Tcells), and primary T cells overexpress CD47 and include a genomicmodification of the B2M gene. In some embodiments, pluripotent stemcells, differentiated cell derived from such pluripotent stem cells andprimary T cells overexpress CD47 and include a genomic modification ofthe CIITA gene. In some embodiments, pluripotent stem cells, T cellsdifferentiated from such pluripotent stem cells and primary T cellsoverexpress CD47 and include a genomic modification of the TRAC gene. Insome embodiments, pluripotent stem cells, T cells differentiated fromsuch pluripotent stem cells and primary T cells overexpress CD47 andinclude a genomic modification of the TRB gene. In some embodiments,pluripotent stem cells, T cells differentiated from such pluripotentstem cells and primary T cells overexpress CD47 and include one or moregenomic modifications selected from the group consisting of the B2M,CIITA, TRAC and TRB genes. In some embodiments, pluripotent stem cells,T cells differentiated from such pluripotent stem cells and primary Tcells overexpress CD47 and include genomic modifications of the B2M,CIITA and TRAC genes. In some embodiments, pluripotent stem cells, Tcells differentiated from such pluripotent stem cells and primary Tcells overexpress CD47 and include genomic modifications of the B2M,CIITA and TRB genes. In some embodiments, pluripotent stem cells, Tcells differentiated from such pluripotent stem cells and primary Tcells overexpress CD47 and include genomic modifications of the B2M,CIITA, TRAC and TRB genes. In certain embodiments, the pluripotent stemcells, differentiated cell derived from such pluripotent stem cells andprimary T cells are B2M^(−/−), CIITA^(−/−), TRAC^(−/−), CD47tg cells. Incertain embodiments, the cells are B2M^(−/−), CIITA^(−/−), TRB^(−/−),CD47tg cells. In certain embodiments, the cells are B2M^(−/−),CIITA^(−/−), TRAC^(−/−), TRB^(−/−), CD47tg cells. In some embodiments,the cells are B2M^(indel/indel), CIITA^(indel/indel),TRAC^(indel/indel), CD47tg cells. In some embodiments, the cells areB2M^(indel/indel), CIITA^(indel/indel), TRB^(indel/indel d)CD47tg cells.In some embodiments, the cells are B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) TRB^(indel/indel) CD47tg cells.In some embodiments, the engineered or modified cells described arepluripotent stem cells, T cells differentiated from such pluripotentstem cells or primary T cells. Non-limiting examples of primary T cellsinclude CD3+ T cells, CD4+ T cells, CD8+ T cells, naïve T cells,regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells,Th9 cells, Th17 cells, T-follicular helper (Tfh) cells, cytotoxic Tlymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm)cells, effector memory T (Tem) cells, effector memory T cells expressCD45RA (TEMRA cells), tissue-resident memory (Trm) cells, virtual memoryT cells, innate memory T cells, memory stem cell (Tsc), γδ T cells, andany other subtype of T cells. In some embodiments, the cells aremodified or engineered as compared to a wild-type or control cell,including an unaltered or unmodified wild-type cell or control cell. Insome embodiments, the wild-type cell or the control cell is a startingmaterial. In some embodiments, the starting material is a primary cellcollected from a donor. In some embodiments, the starting material is aprimary blood cell collected from a donor, e.g., via a leukopak. In someembodiments, the starting material is otherwise modified or engineeredto have altered expression of one or more genes to generate theengineered cell.

In some embodiments, a CD47 transgene is inserted into a pre-selectedlocus of the cell. The pre-selected locus can be a safe harbor or targetlocus. Non-limiting examples of a safe harbor or target locus includes aCCR5 gene locus, a CXCR4 gene locus, a PPP1R12C gene locus, an albumingene locus, a SHS231 gene locus, a CLYBL gene locus, a Rosa gene locus,an F3 (CD142) gene locus, a MICA gene locus, a MICB gene locus, a LRP1(CD91) gene locus, a HMGB1 gene locus, an ABO gene locus, an RHD genelocus, a FUT1 locus, and a KDM5D gene locus. In some embodiments, thepre-selected locus is the TRAC locus. In some embodiments, a CD47transgene is inserted into a safe harbor or target locus (e.g., a CCR5gene locus, a CXCR4 gene locus, a PPP1R12C gene locus, an albumin genelocus, a SHS231 gene locus, a CLYBL gene locus, a Rosa gene locus, an F3(CD142) gene locus, a MICA gene locus, a MICB gene locus, a LRP1 (CD91)gene locus, a HMGB1 gene locus, an ABO gene locus, an RHD gene locus, aFUT1 locus, and a KDM5D gene locus. In certain embodiments, a CD47transgene is inserted into the B2M locus. In certain embodiments, a CD47transgene is inserted into the B2M locus. In certain embodiments, a CD47transgene is inserted into the TRAC locus. In certain embodiments, aCD47 transgene is inserted into the TRB locus. In some embodiments, theCD47 transgene is inserted into a pre-selected locus of the cell,including a safe harbor locus, via viral vectortransduction/integration. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope. In some embodiments, the CD47 transgene isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some instances, expression of a CD47 transgene is controlled by aconstitutive promoter. In other instances, expression of a CD47transgene is controlled by an inducible promoter. In some embodiments,the promoter is an EF1alpha (EF1α) promoter. In some embodiments, thepromoter a CAG promoter.

In yet another embodiment, the present disclosure disclosed herein isdirected to pluripotent stem cells, (e.g., pluripotent stem cells andinduced pluripotent stem cells (iPSCs)), T cells derived from suchpluripotent stem cells (e.g., hypoimmune (HIP) T cells), and primary Tcells that have reduced expression or lack expression of MHC class Iand/or MHC class II human leukocyte antigens and have reduced expressionor lack expression of a T-cell receptor (TCR) complex. In someembodiments, the cells have reduced or lack expression of MHC class Iantigens, MHC class II antigens, and TCR complexes.

In some embodiments, pluripotent stem cells (e.g., iPSCs),differentiated cells derived from such (e.g., T cells differentiatedfrom such), and primary T cells include a genomic modification of theB2M gene. In some embodiments, pluripotent stem cells (e.g., iPSCs),differentiated cells derived from such (e.g., T cells differentiatedfrom such), and primary T cells include a genomic modification of theCIITA gene. In some embodiments, pluripotent stem cells (e.g., iPSCs), Tcells differentiated from such, and primary T cells include a genomicmodification of the TRAC gene. In some embodiments, pluripotent stemcells (e.g., iPSCs), T cells differentiated from such, and primary Tcells include a genomic modification of the TRB gene. In someembodiments, pluripotent stem cells (e.g., iPSCs), T cellsdifferentiated from such, and primary T cells include one or moregenomic modifications selected from the group consisting of the B2M,CIITA and TRAC genes. In some embodiments, pluripotent stem cells (e.g.,iPSCs), T cells differentiated from such, and primary T cells includeone or more genomic modifications selected from the group consisting ofthe B2M, CIITA and TRB genes. In some embodiments, pluripotent stemcells (e.g., iPSCs), T cells differentiated from such, and primary Tcells include one or more genomic modifications selected from the groupconsisting of the B2M, CIITA, TRAC and TRB genes. In certainembodiments, the cells including iPSCs, T cells differentiated fromsuch, and primary T cells are B2M^(−/−), CIITA^(−/−), TRAC-cells. Incertain embodiments, the cells including iPSCs, T cells differentiatedfrom such, and primary T cells are B2M^(−/−), CIITA^(−/−), TRB-cells. Insome embodiments, the cells including iPSCs, T cells differentiated fromsuch, and primary T cells are B2M^(indel/indel), CIITA^(indel/indel),TRAC^(indel/indel) cells. In some embodiments, the cells includingiPSCs, T cells differentiated from such, and primary T cells areB2M^(indel/indel), CIITA^(indel/indel), TRB^(indel/indel) cells. In someembodiments, the cells including iPSCs, T cells differentiated fromsuch, and primary T cells are B2M^(indel/indel), CIITA^(indel/indel),TRAC^(indel/indel), TRB^(indel/indel) cells. In some embodiments, themodified cells described are pluripotent stem cells, induced pluripotentstem cells, T cells differentiated from such pluripotent stem cells andinduced pluripotent stem cells, or primary T cells. Non-limitingexamples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ Tcells, naïve T cells, regulatory T (Treg) cells, non-regulatory T cells,Th1 cells, Th2 cells, Th9 cells, Th17 cells, T-follicular helper (Tfh)cells, cytotoxic T lymphocytes (CTL), effector T (Teff) cells, centralmemory T (Tcm) cells, effector memory T (Tem) cells, effector memory Tcells express CD45RA (TEMRA cells), tissue-resident memory (Trm) cells,virtual memory T cells, innate memory T cells, memory stem cell (Tsc),γδ T cells, and any other subtype of T cells. In some embodiments, thecells are modified or engineered as compared to a wild-type or controlcell, including an unaltered or unmodified wild-type cell or controlcell. In some embodiments, the wild-type cell or the control cell is astarting material. In some embodiments, the starting material is aprimary cell collected from a donor. In some embodiments, the startingmaterial is a primary blood cell collected from a donor, e.g., via aleukopak. In some embodiments, the starting material is otherwisemodified or engineered to have altered expression of one or more genesto generate the engineered cell.

Cells of the present disclosure exhibit reduced or lack expression ofMHC class I antigens, MHC class II antigens, and/or TCR complexes.Reduction of MHC I and/or MHC II expression can be accomplished, forexample, by one or more of the following: (1) targeting the polymorphicHLA alleles (HLA-A, HLA-B, HLA-C) and MHC-II genes directly; (2) removalof B2M, which will prevent surface trafficking of all MHC-I molecules;(3) removal of CIITA, which will prevent surface trafficking of allMHC-II molecules; and/or (4) deletion of components of the MHCenhanceosomes, such as LRC5, RFX5, RFXANK, RFXAP, IRF1, NF-Y (includingNFY-A, NFY-B, NFY-C), and CIITA that are critical for HLA expression.

In some embodiments, HLA expression is interfered with by targetingindividual HLAs (e.g., knocking out, knocking down, or reducingexpression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and/or HLA-DR),targeting transcriptional regulators of HLA expression (e.g., knockingout, knocking down, or reducing expression of NLRC5, CIITA, RFX5, RFXAP,RFXANK, NFY-A, NFY-B, NFY-C and/or IRF-1), blocking surface traffickingof MHC class I molecules (e.g., knocking out, knocking down, or reducingexpression of B2M and/or TAP1), and/or targeting with HLA-Razor (see,e.g., WO2016183041).

In some embodiments, the cells disclosed herein including, but notlimited to, pluripotent stem cells, induced pluripotent stem cells,differentiated cells derived from such stem cells, and primary T cellsdo not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B,HLA-C, HLA-DP, HLA-DQ, and/or HLA-DR) corresponding to MHC-I and/orMHC-II and are thus characterized as being hypoimmunogenic. For example,in certain embodiments, the pluripotent stem cells and inducedpluripotent stem cells disclosed have been modified such that the stemcell or a differentiated stem cell prepared therefrom do not express orexhibit reduced expression of one or more of the following MHC-Imolecules: HLA-A, HLA-B and HLA-C. In some embodiments, one or more ofHLA-A, HLA-B and HLA-C may be “knocked-out” of a cell. A cell that has aknocked-out HLA-A gene, HLA-B gene, and/or HLA-C gene may exhibitreduced or eliminated expression of each knocked-out gene.

In some embodiments, guide RNAs, shRNAs, siRNAs, or miRNAs that allowsimultaneous deletion of all MHC class I alleles by targeting aconserved region in the HLA genes are identified as HLA Razors. In someembodiments, the gRNAs are part of a CRISPR system. In alternativeembodiments, the gRNAs are part of a TALEN system. In some embodiments,an HLA Razor targeting an identified conserved region in HLAs isdescribed in WO2016183041. In some embodiments, multiple HLA Razorstargeting identified conserved regions are utilized. It is generallyunderstood that any guide, siRNA, shRNA, or miRNA molecule that targetsa conserved region in HLAs can act as an HLA Razor.

Methods provided are useful for inactivation or ablation of MHC class Iexpression and/or MHC class II expression in cells such as but notlimited to pluripotent stem cells, differentiated cells, and primary Tcells. In some embodiments, genome editing technologies utilizingrare-cutting endonucleases (e.g., the CRISPR/Cas, TALEN, zinc fingernuclease, meganuclease, and homing endonuclease systems) are also usedto reduce or eliminate expression of genes involved in an immuneresponse (e.g., by deleting genomic DNA of genes involved in an immuneresponse or by insertions of genomic DNA into such genes, such that geneexpression is impacted) in cells. In certain embodiments, genome editingtechnologies or other gene modulation technologies are used to inserttolerance-inducing factors in human cells, rendering them and thedifferentiated cells prepared therefrom hypoimmunogenic cells. As such,the hypoimmunogenic cells have reduced or eliminated expression of MHC Iand MHC II expression. In some embodiments, the cells are nonimmunogenic(e.g., do not induce an innate and/or an adaptive immune response) in arecipient subject.

In some embodiments, the cell includes a modification to increaseexpression of CD47 and one or more factors selected from the groupconsisting of DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C,HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-Inhibitor,IL-10, IL-35, IL-39, FasL, CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16Fc receptor, IL15-RF, and/or Serpinb9.

In some embodiments, the cell comprises a genomic modification of one ormore target polynucleotide sequences that regulate the expression ofeither MHC class I molecules, MHC class II molecules, or MHC class I andMHC class II molecules. In some embodiments, a genetic editing system isused to modify one or more target polynucleotide sequences. In someembodiments, the targeted polynucleotide sequence is one or moreselected from the group including B2M, CIITA, and NLRC5. In someembodiments, the cell comprises a genetic editing modification to theB2M gene. In some embodiments, the cell comprises a genetic editingmodification to the CIITA gene. In some embodiments, the cell comprisesa genetic editing modification to the NLRC5 gene. In some embodiments,the cell comprises genetic editing modifications to the B2M and CIITAgenes. In some embodiments, the cell comprises genetic editingmodifications to the B2M and NLRC5 genes. In some embodiments, the cellcomprises genetic editing modifications to the CIITA and NLRC5 genes. Innumerous embodiments, the cell comprises genetic editing modificationsto the B2M, CIITA and NLRC5 genes. In certain embodiments, the genome ofthe cell has been altered to reduce or delete critical components of HLAexpression. In some embodiments, the cells are modified or engineered ascompared to a wild-type or control cell, including an unaltered orunmodified wild-type cell or control cell. In some embodiments, thewild-type cell or the control cell is a starting material. In someembodiments, the starting material is a primary cell collected from adonor. In some embodiments, the starting material is a primary bloodcell collected from a donor, e.g., via a leukopak. In some embodiments,the starting material is otherwise modified or engineered to havealtered expression of one or more genes to generate the engineered cell.

In some embodiments, the present disclosure provides a cell (e.g., stemcell, induced pluripotent stem cell, differentiated cell such as aprimary NK cell, CAR-NK cell, primary T cell or CAR-T cell) orpopulation thereof comprising a genome in which a gene has been editedto delete a contiguous stretch of genomic DNA, thereby reducing oreliminating surface expression of MHC class I molecules in the cell orpopulation thereof. In certain embodiments, the present disclosureprovides a cell (e.g., stem cell, induced pluripotent stem cell,differentiated cell such as a primary NK cell, CAR-NK cell, primary Tcell or CAR-T cell) or population thereof comprising a genome in which agene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating surface expression of MHC class IImolecules in the cell or population thereof. In numerous embodiments,the present disclosure provides a cell (e.g., stem cell, inducedpluripotent stem cell, differentiated cell, hematopoietic stem cell,primary T cell or CAR-T cell) or population thereof comprising a genomein which one or more genes has been edited to delete a contiguousstretch of genomic DNA, thereby reducing or eliminating surfaceexpression of MHC class I and II molecules in the cell or populationthereof.

In certain embodiments, the expression of MHC I molecules and/or MHC IImolecules is modulated by targeting and deleting a contiguous stretch ofgenomic DNA, thereby reducing or eliminating expression of a target geneselected from the group consisting of B2M, CIITA, and NLRC5. In someembodiments, described herein are genetically edited cells (e.g.,modified human cells) comprising exogenous CD47 proteins and inactivatedor modified CIITA gene sequences, and in some instances, additional genemodifications that inactivate or modify B2M gene sequences. In someembodiments, described herein are genetically edited cells comprisingexogenous CD47 proteins and inactivated or modified CIITA genesequences, and in some instances, additional gene modifications thatinactivate or modify NLRC5 gene sequences. In some embodiments,described herein are genetically edited cells comprising exogenous CD47proteins and inactivated or modified B2M gene sequences, and in someinstances, additional gene modifications that inactivate or modify NLRC5gene sequences. In some embodiments, described herein are geneticallyedited cells comprising exogenous CD47 proteins and inactivated ormodified B2M gene sequences, and in some instances, additional genemodifications that inactivate or modify CIITA gene sequences and NLRC5gene sequences.

Provided herein are cells exhibiting a modification of one or moretargeted polynucleotide sequences that regulates the expression of anyone of the following: (a) MHC I antigens, (b) MHC II antigens, (c) TCRcomplexes, (d) both MHC I and II antigens, and (e) MHC I and II antigensand TCR complexes. In certain embodiments, the modification includesincreasing expression of CD47. In some embodiments, the cells include anexogenous or recombinant CD47 polypeptide. In certain embodiments, themodification includes expression of a chimeric antigen receptor. In someembodiments, the cells comprise an exogenous or recombinant chimericantigen receptor polypeptide.

In some embodiments, the cell includes a genomic modification of one ormore targeted polynucleotide sequences that regulates the expression ofMHC I antigens, MHC II antigens and/or TCR complexes. In someembodiments, a genetic editing system is used to modify one or moretargeted polynucleotide sequences. In some embodiments, thepolynucleotide sequence targets one or more genes selected from thegroup consisting of B2M, CIITA, TRAC, and TRB. In certain embodiments,the genome of a T cell (e.g., a T cell differentiated fromhypoimmunogenic iPSCs and a primary T cell) has been altered to reduceor delete critical components of HLA and TCR expression, e.g., HLA-Aantigen, HLA-B antigen, HLA-C antigen, HLA-DP antigen, HLA-DQ antigen,HLA-DR antigens, TCR-alpha and TCR-beta.

In some embodiments, the present disclosure provides a cell orpopulation thereof comprising a genome in which a gene has been editedto delete a contiguous stretch of genomic DNA, thereby reducing oreliminating surface expression of MHC class I molecules in the cell orpopulation thereof. In certain embodiments, the present disclosureprovides a cell or population thereof comprising a genome in which agene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating surface expression of MHC class IImolecules in the cell or population thereof. In certain embodiments, thepresent disclosure provides a cell or population thereof comprising agenome in which a gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of TCRmolecules in the cell or population thereof. In numerous embodiments,the present disclosure provides a cell or population thereof comprisinga genome in which one or more genes has been edited to delete acontiguous stretch of genomic DNA, thereby reducing or eliminatingsurface expression of MHC class I and II molecules and TCR complexmolecules in the cell or population thereof.

In some embodiments, the cells and methods described herein includegenomically editing human cells to cleave CIITA gene sequences as wellas editing the genome of such cells to alter one or more additionaltarget polynucleotide sequences such as, but not limited to, B2M TRAC,and TRB. In some embodiments, the cells and methods described hereininclude genomically editing human cells to cleave B2M gene sequences aswell as editing the genome of such cells to alter one or more additionaltarget polynucleotide sequences such as, but not limited to, CIITA,TRAC, and TRB. In some embodiments, the cells and methods describedherein include genomically editing human cells to cleave TRAC genesequences as well as editing the genome of such cells to alter one ormore additional target polynucleotide sequences such as, but not limitedto, B2M, CIITA, and TRB. In some embodiments, the cells and methodsdescribed herein include genomically editing human cells to cleave TRBgene sequences as well as editing the genome of such cells to alter oneor more additional target polynucleotide sequences such as, but notlimited to, B2M, CIITA, and TRAC.

Provided herein are hypoimmunogenic stem cells comprising reducedexpression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA,TCR-alpha, and TCR-beta relative to a wild-type stem cell, thehypoimmunogenic stem cell further comprising a set of exogenouspolynucleotides comprising a first exogenous polynucleotide encodingCD47 and a second exogenous polynucleotide encoding a chimeric antigenreceptor (CAR), wherein the first and/or second exogenouspolynucleotides are inserted into a specific locus of at least oneallele of the cell. Also provided herein are hypoimmunogenic primary Tcells including any subtype of primary T cells comprising reducedexpression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA,TCR-alpha, and TCR-beta relative to a wild-type primary T cell, thehypoimmunogenic stem cell further comprising a set of exogenouspolynucleotides comprising a first exogenous polynucleotide encodingCD47 and a second exogenous polynucleotide encoding a chimeric antigenreceptor (CAR), wherein the first and/or second exogenouspolynucleotides are inserted into a specific locus of at least oneallele of the cell. Further provided herein are hypoimmunogenic T cellsdifferentiated from hypoimmunogenic induced pluripotent stem cellscomprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ,HLA-DR, B2M, CIITA, TCR-alpha, and TCR-beta relative to a wild-typeprimary T cell, the hypoimmunogenic stem cell further comprising a setof exogenous polynucleotides comprising a first exogenous polynucleotideencoding CD47 and a second exogenous polynucleotide encoding a chimericantigen receptor (CAR), wherein the first and/or second exogenouspolynucleotides are inserted into a specific locus of at least oneallele of the cell.

In some embodiments, the population of engineered cells described evadesNK cell mediated cytotoxicity upon administration to a recipientpatient. In some embodiments, the population of engineered cells evadesNK cell mediated cytotoxicity by one or more subpopulations of NK cells.In some embodiments, the population of engineered eis protected fromcell lysis by NK cells, including immature and/or mature NK cells uponadministration to a recipient patient. In some embodiments, thepopulation of engineered cells evades macrophage engulfment uponadministration to a recipient patient. In some embodiments, thepopulation of engineered cells does not induce an innate and/or anadaptive immune response to the cell upon administration to a recipientpatient.

In some embodiments, the cells described herein comprise a safetyswitch. The term “safety switch” used herein refers to a system forcontrolling the expression of a gene or protein of interest that, whendownregulated or upregulated, leads to clearance or death of the cell,e.g., through recognition by the host's immune system. A safety switchcan be designed to be triggered by an exogenous molecule in case of anadverse clinical event. A safety switch can be engineered by regulatingthe expression on the DNA, RNA and protein levels. A safety switchincludes a protein or molecule that allows for the control of cellularactivity in response to an adverse event. In one embodiment, the safetyswitch is a “kill switch” that is expressed in an inactive state and isfatal to a cell expressing the safety switch upon activation of theswitch by a selective, externally provided agent. In one embodiment, thesafety switch gene is cis-acting in relation to the gene of interest ina construct. Activation of the safety switch causes the cell to killsolely itself or itself and neighboring cells through apoptosis ornecrosis. In some embodiments, the cells described herein, e.g., stemcells, induced pluripotent stem cells, hematopoietic stem cells, primarycells, or differentiated cell, including, but not limited to, T cells,CAR-T cells, NK cells, and/or CAR-NK cells, comprise a safety switch.

In some embodiments, the safety switch comprises a therapeutic agentthat inhibits or blocks the interaction of CD47 and SIRPα. In someaspects, the CD47-SIRPα blockade agent is an agent that neutralizes,blocks, antagonizes, or interferes with the cell surface expression ofCD47, SIRPα, or both. In some embodiments, the CD47-SIRPα blockade agentinhibits or blocks the interaction of CD47, SIRPα or both. In someembodiments, a CD47-SIRPα blockade agent (e.g., a CD47-SIRPα blocking,inhibiting, reducing, antagonizing, neutralizing, or interfering agent)comprises an agent selected from a group that includes an antibody orfragment thereof that binds CD47, a bispecific antibody that binds CD47,an immunocytokine fusion protein that bind CD47, a CD47 containingfusion protein, an antibody or fragment thereof that binds SIRPα, abispecific antibody that binds SIRPα, an immunocytokine fusion proteinthat bind SIRPα, an SIRPα containing fusion protein, and a combinationthereof.

In some embodiments, the cells described herein comprise a “suicidegene” (or “suicide switch”). The suicide gene can cause the death of thehypoimmunogenic cells should they grow and divide in an undesiredmanner. The suicide gene ablation approach includes a suicide gene in agene transfer vector encoding a protein that results in cell killingonly when activated by a specific compound. A suicide gene can encode anenzyme that selectively converts a nontoxic compound into highly toxicmetabolites. In some embodiments, the cells described herein, e.g., stemcells, induced pluripotent stem cells, hematopoietic stem cells, primarycells, or differentiated cell, including, but not limited to, T cells,CAR-T cells, NK cells, and/or CAR-NK cells, comprise a suicide gene.

In some embodiments, the population of engineered cells describedelicits a reduced level of immune activation or no immune activationupon administration to a recipient subject. In some embodiments, thecells elicit a reduced level of systemic TH1 activation or no systemicTH1 activation in a recipient subject. In some embodiments, the cellselicit a reduced level of immune activation of peripheral bloodmononuclear cells (PBMCs) or no immune activation of PBMCs in arecipient subject. In some embodiments, the cells elicit a reduced levelof donor-specific IgG antibodies or no donor specific IgG antibodiesagainst the cells upon administration to a recipient subject. In someembodiments, the cells elicit a reduced level of IgM and IgG antibodyproduction or no IgM and IgG antibody production against the cells in arecipient subject. In some embodiments, the cells elicit a reduced levelof cytotoxic T cell killing of the cells upon administration to arecipient subject.

B. CIITA

In some embodiments, the technologies disclosed herein modulate (e.g.,reduces or eliminates) the expression of MHC II genes by targeting andmodulating (e.g., reducing or eliminating) Class II transactivator(CIITA) expression. In some embodiments, the modulation occurs using aCRISPR/Cas system. CIITA is a member of the LR or nucleotide bindingdomain (NBD) leucine-rich repeat (LRR) family of proteins and regulatesthe transcription of MHC II by associating with the MHC enhanceosome.

In some embodiments, the target polynucleotide sequence of the presentdisclosure is a variant of CIITA. In some embodiments, the targetpolynucleotide sequence is a homolog of CIITA. In some embodiments, thetarget polynucleotide sequence is an ortholog of CIITA.

In some embodiments, reduced or eliminated expression of CIITA reducesor eliminates expression of one or more of the following MHC class IIare HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

In some embodiments, the cells described herein comprise genemodifications at the gene locus encoding the CIITA protein. In otherwords, the cells comprise a genetic modification at the CIITA locus. Insome instances, the nucleotide sequence encoding the CIITA protein isset forth in RefSeq. No. NM_000246.4 and NCBI Genbank No. U18259. Insome instances, the CIITA gene locus is described in NCBI Gene ID No.4261. In certain cases, the amino acid sequence of CIITA is depicted asNCBI GenBank No. AAA88861.1. Additional descriptions of the CIITAprotein and gene locus can be found in Uniprot No. P33076, HGNC Ref No.7067, and OMIM Ref No. 600005.

In some embodiments, the hypoimmunogenic cells outlined herein comprisea genetic modification targeting the CIITA gene. In some embodiments,the genetic modification targeting the CIITA gene by the rare-cuttingendonuclease comprises a Cas protein or a polynucleotide encoding a Casprotein, and at least one guide ribonucleic acid sequence forspecifically targeting the CIITA gene. In some embodiments, the at leastone guide ribonucleic acid sequence for specifically targeting the CIITAgene is selected from the group consisting of SEQ ID NOS:5184-36352 ofTable 12 of WO2016183041, which is herein incorporated by reference. Insome embodiments, the cell has a reduced ability to induce an innateand/or an adaptive immune response in a recipient subject. In someembodiments, an exogenous nucleic acid encoding a polypeptide asdisclosed herein (e.g., a chimeric antigen receptor, CD47, or anothertolerogenic factor disclosed herein) is inserted at the CIITA gene.

Assays to test whether the CIITA gene has been inactivated are known anddescribed herein. In some embodiments, the resulting geneticmodification of the CIITA gene by PCR and the reduction of HLA-IIexpression can be assays by FACS analysis. In another embodiment, CIITAprotein expression is detected using a Western blot of cells lysatesprobed with antibodies to the CIITA protein. In another embodiment,reverse transcriptase polymerase chain reactions (RT-PCR) are used toconfirm the presence of the inactivating genetic modification. In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction, for example, with a vector.In some embodiments, the vector is a pseudotyped, self-inactivatinglentiviral vector that carries the exogenous polynucleotide. In someembodiments, the vector is a self-inactivating lentiviral vectorpseudotyped with a vesicular stomatitis VSV-G envelope, and whichcarries the exogenous polynucleotide. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using a lentivirus basedviral vector.

C. B2M

In some embodiments, the technologies disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of the accessorychain B2M. In some embodiments, the modulation occurs using a CRISPR/Cassystem. By modulating (e.g., reducing or deleting) expression of B2M,surface trafficking of MHC-I molecules is blocked and the cell renderedhypoimmunogenic. In some embodiments, the cell has a reduced ability toinduce an innate and/or an adaptive immune response in a recipientsubject.

In some embodiments, the target polynucleotide sequence of the presentdisclosure is a variant of B2M. In some embodiments, the targetpolynucleotide sequence is a homolog of B2M. In some embodiments, thetarget polynucleotide sequence is an ortholog of B2M.

In some embodiments, decreased or eliminated expression of B2M reducesor eliminates expression of one or more of the following MHC Imolecules: HLA-A, HLA-B, and HLA-C.

In some embodiments, the cells described herein comprise genemodifications at the gene locus encoding the B2M protein. In otherwords, the cells comprise a genetic modification at the B2M locus. Insome instances, the nucleotide sequence encoding the B2M protein is setforth in RefSeq. No. NM_004048.4 and Genbank No. AB021288.1. In someinstances, the B2M gene locus is described in NCBI Gene ID No. 567. Incertain cases, the amino acid sequence of B2M is depicted as NCBIGenBank No. BAA35182.1. Additional descriptions of the B2M protein andgene locus can be found in Uniprot No. P61769, HGNC Ref. No. 914, andOMIM Ref No. 109700.

In some embodiments, the hypoimmunogenic cells outlined herein comprisea genetic modification targeting the B2M gene. In some embodiments, thegenetic modification targeting the B2M gene by the rare-cuttingendonuclease comprises a Cas protein or a polynucleotide encoding a Casprotein, and at least one guide ribonucleic acid sequence forspecifically targeting the B2M gene. In some embodiments, the at leastone guide ribonucleic acid sequence for specifically targeting the B2Mgene is selected from the group consisting of SEQ ID NOS:81240-85644 ofTable 15 of WO2016183041, which is herein incorporated by reference. Insome embodiments, an exogenous nucleic acid encoding a polypeptide asdisclosed herein (e.g., a chimeric antigen receptor, CD47, or anothertolerogenic factor disclosed herein) is inserted at the B2M gene. Insome embodiments, the exogenous polynucleotide is inserted into at leastone allele of the cell using viral transduction, for example, with avector. In some embodiments, the vector is a pseudotyped,self-inactivating lentiviral vector that carries the exogenouspolynucleotide. In some embodiments, the vector is a self-inactivatinglentiviral vector pseudotyped with a vesicular stomatitis VSV-Genvelope, and which carries the exogenous polynucleotide. In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction. In some embodiments, theexogenous polynucleotide is inserted into at least one allele of thecell using a lentivirus based viral vector.

Assays to test whether the B2M gene has been inactivated are known anddescribed herein. In some embodiments, the resulting geneticmodification of the B2M gene by PCR and the reduction of HLA-Iexpression can be assays by FACS analysis. In another embodiment, B2Mprotein expression is detected using a Western blot of cells lysatesprobed with antibodies to the B2M protein. In another embodiment,reverse transcriptase polymerase chain reactions (RT-PCR) are used toconfirm the presence of the inactivating genetic modification.

D. NLRC5

In many embodiments, the technologies disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of the NLR family,CARD domain containing 5/NOD27/CLR16.1 (NLRC5). In some embodiments, themodulation occurs using a CRISPR/Cas system. NLRC5 is a criticalregulator of MHC-I-mediated immune responses and, similar to CIITA,NLRC5 is highly inducible by IFN-7 and can translocate into the nucleus.NLRC5 activates the promoters of MHC-I genes and induces thetranscription of MHC-I as well as related genes involved in MHC-Iantigen presentation.

In some embodiments, the target polynucleotide sequence is a variant ofNLRC5. In some embodiments, the target polynucleotide sequence is ahomolog of NLRC5. In some embodiments, the target polynucleotidesequence is an ortholog of NLRC5.

In some embodiments, decreased or eliminated expression of NLRC5 reducesor eliminates expression of one or more of the following MHC Imolecules—HLA-A, HLA-B, and HLA-C.

In some embodiments, the cells outlined herein comprise a geneticmodification targeting the NLRC5 gene. In some embodiments, the geneticmodification targeting the NLRC5 gene by the rare-cutting endonucleasecomprises a Cas protein or a polynucleotide encoding a Cas protein, andat least one guide ribonucleic acid sequence for specifically targetingthe NLRC5 gene. In some embodiments, the at least one guide ribonucleicacid sequence for specifically targeting the NLRC5 gene is selected fromthe group consisting of SEQ ID NOS:36353-81239 of Appendix 3 or Table 14of WO2016183041, the disclosure is incorporated by reference in itsentirety.

Assays to test whether the NLRC5 gene has been inactivated are known anddescribed herein. In some embodiments, the resulting geneticmodification of the NLRC5 gene by PCR and the reduction of HLA-Iexpression can be assays by FACS analysis. In another embodiment, NLRC5protein expression is detected using a Western blot of cells lysatesprobed with antibodies to the NLRC5 protein. In another embodiment,reverse transcriptase polymerase chain reactions (RT-PCR) are used toconfirm the presence of the inactivating genetic modification.

E. TRAC

In many embodiments, the technologies disclosed herein modulate (e.g.,reduce or eliminate) the expression of TCR genes including the TRAC geneby targeting and modulating (e.g., reducing or eliminating) expressionof the constant region of the T cell receptor alpha chain. In someembodiments, the modulation occurs using a CRISPR/Cas system. Bymodulating (e.g., reducing or deleting) expression of TRAC, surfacetrafficking of TCR molecules is blocked. In some embodiments, the cellalso has a reduced ability to induce an innate and/or an adaptive immuneresponse in a recipient subject.

In some embodiments, the target polynucleotide sequence of the presentdisclosure is a variant of TRAC. In some embodiments, the targetpolynucleotide sequence is a homolog of TRAC. In some embodiments, thetarget polynucleotide sequence is an ortholog of TRAC.

In some embodiments, decreased or eliminated expression of TRAC reducesor eliminates TCR surface expression.

In some embodiments, the cells, such as, but not limited to, pluripotentstem cells, induced pluripotent stem cells, T cells differentiated frominduced pluripotent stem cells, primary T cells, and cells derived fromprimary T cells comprise gene modifications at the gene locus encodingthe TRAC protein. In other words, the cells comprise a geneticmodification at the TRAC locus. In some instances, the nucleotidesequence encoding the TRAC protein is set forth in Genbank No. X02592.1.In some instances, the TRAC gene locus is described in RefSeq. No.NG_001332.3 and NCBI Gene ID No. 28755. In certain cases, the amino acidsequence of TRAC is depicted as Uniprot No. P01848. Additionaldescriptions of the TRAC protein and gene locus can be found in UniprotNo. P01848, HGNC Ref No. 12029, and OMIM Ref. No. 186880.

In some embodiments, the hypoimmunogenic cells outlined herein comprisea genetic modification targeting the TRAC gene. In some embodiments, thegenetic modification targeting the TRAC gene by the rare-cuttingendonuclease comprises a Cas protein or a polynucleotide encoding a Casprotein, and at least one guide ribonucleic acid sequence forspecifically targeting the TRAC gene. In some embodiments, the at leastone guide ribonucleic acid sequence for specifically targeting the TRACgene is selected from the group consisting of SEQ ID NOS:532-609 and9102-9797 of US20160348073, which is herein incorporated by reference.

Assays to test whether the TRAC gene has been inactivated are known anddescribed herein. In some embodiments, the resulting geneticmodification of the TRAC gene by PCR and the reduction of TCR expressioncan be assays by FACS analysis. In another embodiment, TRAC proteinexpression is detected using a Western blot of cells lysates probed withantibodies to the TRAC protein. In another embodiment, reversetranscriptase polymerase chain reactions (RT-PCR) are used to confirmthe presence of the inactivating genetic modification.

F. TRB

In many embodiments, the technologies disclosed herein modulate (e.g.,reduce or eliminate) the expression of TCR genes including the geneencoding T cell antigen receptor, beta chain (e.g., the TRB, TRBC, orTCRB gene) by targeting and modulating (e.g., reducing or eliminating)expression of the constant region of the T cell receptor beta chain. Insome embodiments, the modulation occurs using a CRISPR/Cas system. Bymodulating (e.g., reducing or deleting) expression of TRB, surfacetrafficking of TCR molecules is blocked. In some embodiments, the cellalso has a reduced ability to induce an innate and/or an adaptive immuneresponse in a recipient subject.

In some embodiments, the target polynucleotide sequence of the presentdisclosure is a variant of TRB. In some embodiments, the targetpolynucleotide sequence is a homolog of TRB. In some embodiments, thetarget polynucleotide sequence is an ortholog of TRB.

In some embodiments, decreased or eliminated expression of TRB reducesor eliminates TCR surface expression.

In some embodiments, the cells, such as, but not limited to, pluripotentstem cells, induced pluripotent stem cells, T cells differentiated frominduced pluripotent stem cells, primary T cells, and cells derived fromprimary T cells comprise gene modifications at the gene locus encodingthe TRB protein. In other words, the cells comprise a geneticmodification at the TRB gene locus. In some instances, the nucleotidesequence encoding the TRB protein is set forth in UniProt No. P0DSE2. Insome instances, the TRB gene locus is described in RefSeq. No.NG_001333.2 and NCBI Gene ID No. 6957. In certain cases, the amino acidsequence of TRB is depicted as Uniprot No. P01848. Additionaldescriptions of the TRB protein and gene locus can be found in GenBankNo. L36092.2, Uniprot No. P0DSE2, and HGNC Ref. No. 12155.

In some embodiments, the hypoimmunogenic cells outlined herein comprisea genetic modification targeting the TRB gene. In some embodiments, thegenetic modification targeting the TRB gene by the rare-cuttingendonuclease comprises a Cas protein or a polynucleotide encoding a Casprotein, and at least one guide ribonucleic acid sequence forspecifically targeting the TRB gene. In some embodiments, the at leastone guide ribonucleic acid sequence for specifically targeting the TRBgene is selected from the group consisting of SEQ ID NOS:610-765 and9798-10532 of US20160348073, which is herein incorporated by reference.

Assays to test whether the TRB gene has been inactivated are known anddescribed herein. In some embodiments, the resulting geneticmodification of the TRB gene by PCR and the reduction of TCR expressioncan be assays by FACS analysis. In another embodiment, TRB proteinexpression is detected using a Western blot of cells lysates probed withantibodies to the TRB protein. In another embodiment, reversetranscriptase polymerase chain reactions (RT-PCR) are used to confirmthe presence of the inactivating genetic modification.

G. CD142

In many embodiments, the technologies disclosed herein modulate (e.g.,reduce or eliminate) the expression of CD142, which is also known astissue factor, factor III, and F3. In some embodiments, the modulationoccurs using a gene editing system (e.g., CRISPR/Cas).

In some embodiments, the target polynucleotide sequence is CD142 or avariant of CD142. In some embodiments, the target polynucleotidesequence is a homolog of CD142. In some embodiments, the targetpolynucleotide sequence is an ortholog of CD142.

In some embodiments, the cells outlined herein comprise a geneticmodification targeting the CD142 gene. In some embodiments, the geneticmodification targeting the CD142 gene by the rare-cutting endonucleasecomprises a Cas protein or a polynucleotide encoding a Cas protein, andat least one guide ribonucleic acid (gRNA) sequence for specificallytargeting the CD142 gene. Useful methods for identifying gRNA sequencesto target CD142 are described below.

Assays to test whether the CD142 gene has been inactivated are known anddescribed herein. In some embodiments, the resulting geneticmodification of the CD142 gene by PCR and the reduction of CD142expression can be assays by FACS analysis. In another embodiment, CD142protein expression is detected using a Western blot of cells lysatesprobed with antibodies to the CD142 protein. In another embodiment,reverse transcriptase polymerase chain reactions (RT-PCR) are used toconfirm the presence of the inactivating genetic modification.

Useful genomic, polynucleotide and polypeptide information about thehuman CD142 are provided in, for example, the GeneCard IdentifierGC01M094530, HGNC No. 3541, NCBI Gene ID 2152, NCBI RefSeq Nos.NM_001178096.1, NM_001993.4, NP_001171567.1, and NP_001984.1, UniProtNo. P13726, and the like.

H. CTLA-4

In some embodiments, the target polynucleotide sequence is CTLA-4 or avariant of CTLA-4. In some embodiments, the target polynucleotidesequence is a homolog of CTLA-4. In some embodiments, the targetpolynucleotide sequence is an ortholog of CTLA-4.

In some embodiments, the cells outlined herein comprise a geneticmodification targeting the CTLA-4 gene. In certain embodiments, primaryT cells comprise a genetic modification targeting the CTLA-4 gene. Thegenetic modification can reduce expression of CTLA-4 polynucleotides andCTLA-4 polypeptides in T cells includes primary T cells and CAR-T cells.In some embodiments, the genetic modification targeting the CTLA-4 geneby the rare-cutting endonuclease comprises a Cas protein or apolynucleotide encoding a Cas protein, and at least one guideribonucleic acid (gRNA) sequence for specifically targeting the CTLA-4gene. Useful methods for identifying gRNA sequences to target CTLA-4 aredescribed below.

Assays to test whether the CTLA-4 gene has been inactivated are knownand described herein. In some embodiments, the resulting geneticmodification of the CTLA-4 gene by PCR and the reduction of CTLA-4expression can be assays by FACS analysis. In another embodiment, CTLA-4protein expression is detected using a Western blot of cells lysatesprobed with antibodies to the CTLA-4 protein. In another embodiment,reverse transcriptase polymerase chain reactions (RT-PCR) are used toconfirm the presence of the inactivating genetic modification.

Useful genomic, polynucleotide and polypeptide information about thehuman CTLA-4 are provided in, for example, the GeneCard IdentifierGC02P203867, HGNC No. 2505, NCBI Gene ID 1493, NCBI RefSeq Nos.NM_005214.4, NM_001037631.2, NP_001032720.1 and NP_005205.2, UniProt No.P16410, and the like.

I. PD-1

In some embodiments, the target polynucleotide sequence is PD-1 or avariant of PD-1. In some embodiments, the target polynucleotide sequenceis a homolog of PD-1. In some embodiments, the target polynucleotidesequence is an ortholog of PD-1.

In some embodiments, the cells outlined herein comprise a geneticmodification targeting the gene encoding the programmed cell deathprotein 1 (PD-1) protein or the PDCD1 gene. In certain embodiments,primary T cells comprise a genetic modification targeting the PDCD1gene. The genetic modification can reduce expression of PD-1polynucleotides and PD-1 polypeptides in T cells includes primary Tcells and CAR-T cells. In some embodiments, the genetic modificationtargeting the PDCD1 gene by the rare-cutting endonuclease comprises aCas protein or a polynucleotide encoding a Cas protein, and at least oneguide ribonucleic acid (gRNA) sequence for specifically targeting thePDCD1 gene. Useful methods for identifying gRNA sequences to target PD-1are described below.

Assays to test whether the PDCD1 gene has been inactivated are known anddescribed herein. In some embodiments, the resulting geneticmodification of the PDCD1 gene by PCR and the reduction of PD-1expression can be assays by FACS analysis. In another embodiment, PD-1protein expression is detected using a Western blot of cells lysatesprobed with antibodies to the PD-1 protein. In another embodiment,reverse transcriptase polymerase chain reactions (RT-PCR) are used toconfirm the presence of the inactivating genetic modification.

Useful genomic, polynucleotide and polypeptide information about humanPD-1 including the PDCD1 gene are provided in, for example, the GeneCardIdentifier GC02M241849, HGNC No. 8760, NCBI Gene ID 5133, Uniprot No.Q15116, and NCBI RefSeq Nos. NM_005018.2 and NP_005009.2.

J. CD47

In some embodiments, the present disclosure provides a cell orpopulation thereof that has been modified to express the tolerogenicfactor (e.g., immunomodulatory polypeptide) CD47. In some embodiments,the present disclosure provides a method for altering a cell genome toexpress CD47. In some embodiments, the stem cell expresses exogenousCD47. In some instances, the cell expresses an expression vectorcomprising a nucleotide sequence encoding a human CD47 polypeptide. Insome embodiments, the cell is genetically modified to comprise anintegrated exogenous polynucleotide encoding CD47 usinghomology-directed repair. In some instances, the cell expresses anucleotide sequence encoding a human CD47 polypeptide such that thenucleotide sequence is inserted into at least one allele of a safeharbor or target locus. In some instances, the cell expresses anucleotide sequence encoding a human CD47 polypeptide wherein thenucleotide sequence is inserted into at least one allele of an AAVS1locus. In some instances, the cell expresses a nucleotide sequenceencoding a human CD47 polypeptide wherein the nucleotide sequence isinserted into at least one allele of an CCR5 locus. In some instances,the cell expresses a nucleotide sequence encoding a human CD47polypeptide wherein the nucleotide sequence is inserted into at leastone allele of a safe harbor or target gene locus, such as, but notlimited to, a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C genelocus, an albumin gene locus, a SHS231 gene locus, a CLYBL gene locus, aRosa gene locus, an F3 (CD142) gene locus, a MICA gene locus, a MICBgene locus, a LRP1 (CD91) gene locus, a HMGB1 gene locus, an ABO genelocus, an RHD gene locus, a FUT1 locus, and a KDM5D gene locus. In someinstances, the cell expresses a nucleotide sequence encoding a humanCD47 polypeptide wherein the nucleotide sequence is inserted into atleast one allele of a TRAC locus.

CD47 is a leukocyte surface antigen and has a role in cell adhesion andmodulation of integrins. It is expressed on the surface of a cell andsignals to circulating macrophages not to eat the cell.

In some embodiments, the cell outlined herein comprises a nucleotidesequence encoding a CD47 polypeptide has at least 95% sequence identity(e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence asset forth in NCBI Ref Sequence Nos. NP_001768.1 and NP_942088.1. In someembodiments, the cell outlined herein comprises a nucleotide sequenceencoding a CD47 polypeptide having an amino acid sequence as set forthin NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1. In someembodiments, the cell comprises a nucleotide sequence for CD47 having atleast 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence setforth in NCBI Ref Nos. NM_001777.3 and NM_198793.2. In some embodiments,the cell comprises a nucleotide sequence for CD47 as set forth in NCBIRef. Sequence Nos. NM_001777.3 and NM_198793.2. In some embodiments, thenucleotide sequence encoding a CD47 polynucleotide is a codon optimizedsequence. In some embodiments, the nucleotide sequence encoding a CD47polynucleotide is a human codon optimized sequence.

In some embodiments, the cell comprises a CD47 polypeptide having atleast 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) toan amino acid sequence as set forth in NCBI Ref Sequence Nos.NP_001768.1 and NP_942088.1. In some embodiments, the cell outlinedherein comprises a CD47 polypeptide having an amino acid sequence as setforth in NCBI Ref Sequence Nos. NP_001768.1 and NP_942088.1.

Exemplary amino acid sequences of human CD47 with a signal sequence andwithout a signal sequence are provided in Table 1.

TABLE 1 Amino acid sequences of human CD47 SEQ Amino acid Protein ID NO:Sequence residues Human 13 QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRaa 19-323 CD47 DIYTFDGALNKSTVPTDFSSAKIEVSQQLLKGDASLKMDKSDAVS (withoutHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFA signalILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILF sequence)VPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE Human 14MWPLVAALLLGSACCGAQLLFNKTKSVEFTFCNDTVVIPCFVTNM aa 1-323 CD47 (withEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQL signalLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSW sequence)FSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSVFIALILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKE SKGMMNDE

In some embodiments, the cell comprises a CD47 polypeptide having atleast 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) tothe amino acid sequence of SEQ ID NO:13. In some embodiments, the cellcomprises a CD47 polypeptide having the amino acid sequence of SEQ IDNO:13. In some embodiments, the cell comprises a CD47 polypeptide havingat least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more)to the amino acid sequence of SEQ ID NO:14. In some embodiments, thecell comprises a CD47 polypeptide having the amino acid sequence of SEQID NO:14.

In some embodiments, the cell comprises a nucleotide sequence encoding aCD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%,97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO:13. Insome embodiments, the cell comprises a nucleotide sequence encoding aCD47 polypeptide having the amino acid sequence of SEQ ID NO:13. In someembodiments, the cell comprises a nucleotide sequence encoding a CD47polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%,98%, 99%, or more) to the amino acid sequence of SEQ ID NO:14. In someembodiments, the cell comprises a nucleotide sequence encoding a CD47polypeptide having the amino acid sequence of SEQ ID NO:14. In someembodiments, the nucleotide sequence is codon optimized for expressionin a particular cell.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cassystem or any of the gene editing systems described herein) is used tofacilitate the insertion of a polynucleotide encoding CD47, into agenomic locus of the hypoimmunogenic cell. In some cases, thepolynucleotide encoding CD47 is inserted into a safe harbor or targetlocus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26,SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, orKDM5D gene locus. In some embodiments, the polynucleotide encoding CD47is inserted into a B2M gene locus, a CIITA gene locus, a TRAC genelocus, or a TRB gene locus. In some embodiments, the polynucleotideencoding CD47 is inserted into any one of the gene loci depicted inTable 15 provided herein. In certain embodiments, the polynucleotideencoding CD47 is operably linked to a promoter.

In some embodiments, the polynucleotide encoding CD47 is inserted intoat least one allele of the T cell using viral transduction. In someembodiments, the polynucleotide encoding CD47 is inserted into at leastone allele of the T cell using a lentivirus based viral vector. In someembodiments, the lentivirus based viral vector is a pseudotyped,self-inactivating lentiviral vector that carries the polynucleotideencoding CD47. In some embodiments, the lentivirus based viral vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the polynucleotide encodingCD47.

In another embodiment, CD47 protein expression is detected using aWestern blot of cell lysates probed with antibodies against the CD47protein. In another embodiment, reverse transcriptase polymerase chainreactions (RT-PCR) are used to confirm the presence of the exogenousCD47 mRNA.

K. CD24

In some embodiments, the present disclosure provides a cell orpopulation thereof that has been modified to express the tolerogenicfactor (e.g., immunomodulatory polypeptide) CD24. In some embodiments,the present disclosure provides a method for altering a cell genome toexpress CD24. In some embodiments, the stem cell expresses exogenousCD24. In some instances, the cell expresses an expression vectorcomprising a nucleotide sequence encoding a human CD24 polypeptide. Insome embodiments, the exogenous polynucleotide is inserted into at leastone allele of the cell using viral transduction, for example, with avector. In some embodiments, the vector is a pseudotyped,self-inactivating lentiviral vector that carries the exogenouspolynucleotide. In some embodiments, the vector is a self-inactivatinglentiviral vector pseudotyped with a vesicular stomatitis VSV-Genvelope, and which carries the exogenous polynucleotide. In someembodiments, the exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction. In some embodiments, theexogenous polynucleotide is inserted into at least one allele of thecell using a lentivirus based viral vector.

CD24 which is also referred to as a heat stable antigen or small-celllung cancer cluster 4 antigen is a glycosylatedglycosylphosphatidylinositol-anchored surface protein (Pirruccello etal., J Immunol, 1986, 136, 3779-3784; Chen et al., Glycobiology, 2017,57, 800-806). It binds to Siglec-10 on innate immune cells. Recently ithas been shown that CD24 via Siglec-10 acts as an innate immunecheckpoint (Barkal et al., Nature, 2019, 572, 392-396).

In some embodiments, the cell outlined herein comprises a nucleotidesequence encoding a CD24 polypeptide has at least 95% sequence identity(e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence setforth in NCBI Ref Nos. NP_001278666.1, NP 001278667.1, NP_001278668.1,and NP_037362.1. In some embodiments, the cell outlined herein comprisesa nucleotide sequence encoding a CD24 polypeptide having an amino acidsequence set forth in NCBI Ref. Nos. NP_001278666.1, NP_001278667.1,NP_001278668.1, and NP_037362.1.

In some embodiments, the cell comprises a nucleotide sequence having atleast 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence setforth in NCBI Ref. Nos. NM_00129737.1, NM 00129738.1, NM_001291739.1,and NM_013230.3. In some embodiments, the cell comprises a nucleotidesequence as set forth in NCBI Ref. Nos. NM_00129737.1, NM_00129738.1,NM_001291739.1, and NM_013230.3.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cassystem or any of the gene editing systems described herein) is used tofacilitate the insertion of a polynucleotide encoding CD24, into agenomic locus of the hypoimmunogenic cell. In some cases, thepolynucleotide encoding CD24 is inserted into a safe harbor or targetlocus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26,SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, orKDM5D gene locus. In some embodiments, the polynucleotide encoding CD24is inserted into a B2M gene locus, a CIITA gene locus, a TRAC genelocus, or a TRB gene locus. In some embodiments, the polynucleotideencoding CD24 is inserted into any one of the gene loci depicted inTable 15 provided herein. In certain embodiments, the polynucleotideencoding CD24 is operably linked to a promoter.

In another embodiment, CD24 protein expression is detected using aWestern blot of cells lysates probed with antibodies against the CD24protein. In another embodiment, reverse transcriptase polymerase chainreactions (RT-PCR) are used to confirm the presence of the exogenousCD24 mRNA.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cassystem or any of the gene editing systems described herein) is used tofacilitate the insertion of a polynucleotide encoding CD24, into agenomic locus of the hypoimmunogenic cell. In some cases, thepolynucleotide encoding CD24 is inserted into a safe harbor or targetlocus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26,SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91),HMGB1, ABO, RHD, FUT1, or KDM5D gene locus. In some embodiments, thepolynucleotide encoding CD24 is inserted into a B2M gene locus, a CIITAgene locus, a TRAC gene locus, or a TRB gene locus. In some embodiments,the polynucleotide encoding CD24 is inserted into any one of the geneloci depicted in Table 15 provided herein. In certain embodiments, thepolynucleotide encoding CD24 is operably linked to a promoter.

L. DUX4

In some embodiments, the present disclosure provides a cell (e.g., stemcell, induced pluripotent stem cell, differentiated cell, hematopoieticstem cell, primary T cell or CAR-T cell) or population thereofcomprising a genome modified to increase expression of a tolerogenic orimmunosuppressive factor such as DUX4. In some embodiments, the presentdisclosure provides a method for altering a cell's genome to provideincreased expression of DUX4, including through a exogenouspolynucleotide. In some embodiments, the disclosure provides a cell orpopulation thereof comprising exogenously expressed DUX4 proteins. Insome embodiments, increased expression of DUX4 suppresses, reduces oreliminates expression of one or more of the following MHC Imolecules—HLA-A, HLA-B, and HLA-C. In some embodiments, the exogenouspolynucleotide is inserted into at least one allele of the cell usingviral transduction, for example, with a vector. In some embodiments, thevector is a pseudotyped, self-inactivating lentiviral vector thatcarries the exogenous polynucleotide. In some embodiments, the vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

DUX4 is a transcription factor that is active in embryonic tissues andinduced pluripotent stem cells, and is silent in normal, healthy somatictissues (Feng et al., 2015, ELife4; De Iaco et al., 2017, Nat Genet, 49,941-945; Hendrickson et al., 2017, Nat Genet, 49, 925-934; Snider etal., 2010, PLoS Genet, e1001181; Whiddon et al., 2017, Nat Genet). DUX4expression acts to block IFN-gamma mediated induction of majorhistocompatibility complex (MHC) class I gene expression (e.g.,expression of B2M, HLA-A, HLA-B, and HLA-C). DUX4 expression has beenimplicated in suppressed antigen presentation by MHC class I (Chew etal., Developmental Cell, 2019, 50, 1-14). DUX4 functions as atranscription factor in the cleavage-stage gene expression(transcriptional) program. Its target genes include, but are not limitedto, coding genes, noncoding genes, and repetitive elements.

There are at least two isoforms of DUX4, with the longest isoformcomprising the DUX4 C-terminal transcription activation domain. Theisoforms are produced by alternative splicing. See, e.g., Geng et al.,2012, Dev Cell, 22, 38-51; Snider et al., 2010, PLoS Genet, e1001181.Active isoforms for DUX4 comprise its N-terminal DNA-binding domains andits C-terminal activation domain. See, e.g., Choi et al., 2016, NucleicAcid Res, 44, 5161-5173.

It has been shown that reducing the number of CpG motifs of DUX4decreases silencing of a DUX4 transgene (Jagannathan et al., HumanMolecular Genetics, 2016, 25(20):4419-4431). The nucleic acid sequenceprovided in Jagannathan et al., supra represents a codon alteredsequence of DUX4 comprising one or more base substitutions to reduce thetotal number of CpG sites while preserving the DUX4 protein sequence.The nucleic acid sequence is commercially available from Addgene,Catalog No. 99281.

In many embodiments, at least one or more polynucleotides may beutilized to facilitate the exogenous expression of DUX4 by a cell, e.g.,a stem cell, induced pluripotent stem cell, differentiated cell,hematopoietic stem cell, primary T cell or CAR-T cell.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cassystem or any of the gene editing systems described herein) is used tofacilitate the insertion of a polynucleotide encoding DUX4, into agenomic locus of the hypoimmunogenic cell. In some cases, thepolynucleotide encoding DUX4 is inserted into a safe harbor or targetlocus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26,SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, orKDM5D gene locus. In some embodiments, the polynucleotide encoding DUX4is inserted into a B2M gene locus, a CIITA gene locus, a TRAC genelocus, or a TRB gene locus. In some embodiments, the polynucleotideencoding DUX4 is inserted into any one of the gene loci depicted inTable 15 provided herein. In certain embodiments, the polynucleotideencoding DUX4 is operably linked to a promoter.

In some embodiments, the polynucleotide encoding DUX4 is inserted intoat least one allele of the T cell using viral transduction. In someembodiments, the polynucleotide encoding DUX4 is inserted into at leastone allele of the T cell using a lentivirus based viral vector. In someembodiments, the lentivirus based viral vector is a pseudotyped,self-inactivating lentiviral vector that carries the polynucleotideencoding DUX4. In some embodiments, the lentivirus based viral vector isa self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the polynucleotide encodingDUX4.

In some embodiments, the polynucleotide sequence encoding DUX4 comprisesa polynucleotide sequence comprising a codon altered nucleotide sequenceof DUX4 comprising one or more base substitutions to reduce the totalnumber of CpG sites while preserving the DUX4 protein sequence. In someembodiments, the polynucleotide sequence encoding DUX4 comprising one ormore base substitutions to reduce the total number of CpG sites has atleast 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:1 ofPCT/US2020/44635, filed Jul. 31, 2020. In some embodiments, thepolynucleotide sequence encoding DUX4 is SEQ ID NO:1 ofPCT/US2020/44635.

In some embodiments, the polynucleotide sequence encoding DUX4 is anucleotide sequence encoding a polypeptide sequence having at least 95%(e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a sequenceselected from a group including SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, asprovided in PCT/US2020/44635. In some embodiments, the polynucleotidesequence encoding DUX4 is a nucleotide sequence encoding a polypeptidesequence is selected from a group including SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQID NO:29. Amino acid sequences set forth as SEQ ID NOS:2-29 are shown inFIG. 1A-1G of PCT/US2020/44635.

In some instances, the DUX4 polypeptide comprises an amino acid sequencehaving at least 95% sequence identity to the sequence set forth inGenBank Accession No. ACN62209.1 or an amino acid sequence set forth inGenBank Accession No. ACN62209.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in NCBI RefSeq No.NP_001280727.1 or an amino acid sequence set forth in NCBI RefSeq No.NP_001280727.1. In some instances, the DUX4 polypeptide comprises anamino acid sequence having at least 95% sequence identity to thesequence set forth in GenBank Accession No. ACP30489.1 or an amino acidsequence set forth in GenBank Accession No. ACP30489.1. In someinstances, the DUX4 polypeptide comprises an amino acid sequence havingat least 95% sequence identity to the sequence set forth in UniProt No.P0CJ85.1 or an amino acid sequence set forth in UniProt No. P0CJ85.1. Insome instances, the DUX4 polypeptide comprises an amino acid sequencehaving at least 95% sequence identity to the sequence set forth inGenBank Accession No. AUA60622.1 or an amino acid sequence set forth inGenBank Accession No. AUA60622.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ADK24683.1 or an amino acid sequence set forth in GenBank Accession No.ADK24683.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in GenBank Accession No. ACN62210.1 or an amino acid sequence setforth in GenBank Accession No. ACN62210.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ADK24706.1 or an amino acid sequence set forth in GenBank Accession No.ADK24706.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in GenBank Accession No. ADK24685.1 or an amino acid sequence setforth in GenBank Accession No. ADK24685.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ACP30488.1 or an amino acid sequence set forth in GenBank Accession No.ACP30488.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in GenBank Accession No. ADK24687.1 or an amino acid sequence setforth in GenBank Accession No. ADK24687.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ACP30487.1 or an amino acid sequence set forth in GenBank Accession No.ACP30487.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in GenBank Accession No. ADK24717.1 or an amino acid sequence setforth in GenBank Accession No. ADK24717.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ADK24690.1 or an amino acid sequence set forth in GenBank Accession No.ADK24690.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in GenBank Accession No. ADK24689.1 or an amino acid sequence setforth in GenBank Accession No. ADK24689.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ADK24692.1 or an amino acid sequence set forth in GenBank Accession No.ADK24692.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in GenBank Accession No. ADK24693.1 or an amino acid sequence ofset forth in GenBank Accession No. ADK24693.1. In some instances, theDUX4 polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ADK24712.1 or an amino acid sequence set forth in GenBank Accession No.ADK24712.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in GenBank Accession No. ADK24691.1 or an amino acid sequence setforth in GenBank Accession No. ADK24691.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in UniProt No. P0CJ87.1 oran amino acid sequence of set forth in UniProt No. P0CJ87.1. In someinstances, the DUX4 polypeptide comprises an amino acid sequence havingat least 95% sequence identity to the sequence set forth in GenBankAccession No. ADK24714.1 or an amino acid sequence set forth in GenBankAccession No. ADK24714.1. In some instances, the DUX4 polypeptidecomprises an amino acid sequence having at least 95% sequence identityto the sequence set forth in GenBank Accession No. ADK24684.1 or anamino acid sequence of set forth in GenBank Accession No. ADK24684.1. Insome instances, the DUX4 polypeptide comprises an amino acid sequencehaving at least 95% sequence identity to the sequence set forth inGenBank Accession No. ADK24695.1 or an amino acid sequence set forth inGenBank Accession No. ADK24695.1. In some instances, the DUX4polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in GenBank Accession No.ADK24699.1 or an amino acid sequence set forth in GenBank Accession No.ADK24699.1. In some instances, the DUX4 polypeptide comprises an aminoacid sequence having at least 95% sequence identity to the sequence setforth in NCBI RefSeq No. NP_001768.1 or an amino acid sequence set forthin NCBI RefSeq No. NP_001768. In some instances, the DUX4 polypeptidecomprises an amino acid sequence having at least 95% sequence identityto the sequence set forth in NCBI RefSeq No. NP_942088.1 or an aminoacid sequence set forth in NCBI RefSeq No. NP_942088.1. In someinstances, the DUX4 polypeptide comprises an amino acid sequence havingat least 95% sequence identity to SEQ ID NO:28 provided inPCT/US2020/44635 or an amino acid sequence of SEQ ID NO:28 provided inPCT/US2020/44635. In some instances, the DUX4 polypeptide comprises anamino acid sequence having at least 95% sequence identity to SEQ IDNO:29 provided in PCT/US2020/44635 or an amino acid sequence of SEQ IDNO:29 provided in PCT/US2020/44635.

In other embodiments, expression of tolerogenic factors is facilitatedusing an expression vector. In some embodiments, the expression vectorcomprises a polynucleotide sequence encoding DUX4 is a codon alteredsequence comprising one or more base substitutions to reduce the totalnumber of CpG sites while preserving the DUX4 protein sequence. In somecases, the codon altered sequence of DUX4 comprises SEQ ID NO:1 ofPCT/US2020/44635. In some cases, the codon altered sequence of DUX4 isSEQ ID NO:1 of PCT/US2020/44635. In other embodiments, the expressionvector comprises a polynucleotide sequence encoding DUX4 comprising SEQID NO:1 of PCT/US2020/44635. In some embodiments, the expression vectorcomprises a polynucleotide sequence encoding a DUX4 polypeptide sequencehaving at least 95% sequence identity to a sequence selected from agroup including SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 of PCT/US2020/44635.In some embodiments, the expression vector comprises a polynucleotidesequence encoding a DUX4 polypeptide sequence selected from a groupincluding SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 of PCT/US2020/44635.

An increase of DUX4 expression can be assayed using known techniques,such as Western blots, ELISA assays, FACS assays, immunoassays, and thelike.

M. Additional Tolerogenic Factors

In many embodiments, one or more tolerogenic factors can be inserted orreinserted into genome-edited cells to create immune-privilegeduniversal donor cells, such as universal donor stem cells, universaldonor T cells, or universal donor cells. In certain embodiments, thehypoimmunogenic cells disclosed herein have been further modified toexpress one or more tolerogenic factors. Exemplary tolerogenic factorsinclude, without limitation, one or more of CD47, DUX4, CD24, CD27,CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G,PD-L1, IDO1, CTLA4-Ig, C1-Inhibitor, IL-10, IL-35, FasL, CCL21, CCL22,Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, and Serpinb9. Insome embodiments, the tolerogenic factors are selected from the groupconsisting of CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1,IDO1, CTLA4-Ig, IL-10, IL-35, FasL, Serpinb9, CCL21, CCL22, and Mfge8.In some embodiments, the tolerogenic factors are selected from the groupconsisting of DUX4, HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig,C1-inhibitor, and IL-35. In some embodiments, the tolerogenic factorsare selected from the group consisting of HLA-C, HLA-E, HLA-F, HLA-G,PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35. In some embodiments, thetolerogenic factors are selected from a group including CD47, DUX4,CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavychain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-Inhibitor, IL-10, IL-35, FasL,CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, andSerpinb9.

In some embodiments, the polynucleotide encoding the one or moretolerogenic factors is inserted into at least one allele of the T cellusing viral transduction. In some embodiments, the polynucleotideencoding the one or more tolerogenic factors is inserted into at leastone allele of the T cell using a lentivirus based viral vector. In someembodiments, the lentivirus based viral vector is a pseudotyped,self-inactivating lentiviral vector that carries the polynucleotideencoding the one or more tolerogenic factors. In some embodiments, thelentivirus based viral vector is a self-inactivating lentiviral vectorpseudotyped with a vesicular stomatitis VSV-G envelope, and whichcarries the polynucleotide encoding the one or more tolerogenic factors.

Useful genomic, polynucleotide and polypeptide information about humanCD27 (which is also known as CD27L receptor, Tumor Necrosis FactorReceptor Superfamily Member 7, TNFSF7, T Cell Activation Antigen 5152,Tp55, and T14) are provided in, for example, the GeneCard IdentifierGC12P008144, HGNC No. 11922, NCBI Gene ID 939, Uniprot No. P26842, andNCBI RefSeq Nos. NM_001242.4 and NP_001233.1.

Useful genomic, polynucleotide and polypeptide information about humanCD46 are provided in, for example, the GeneCard Identifier GC01P207752,HGNC No. 6953, NCBI Gene ID 4179, Uniprot No. P15529, and NCBI RefSeqNos. NM_002389.4, NM_153826.3, NM_172350.2, NM_172351.2, NM_172352.2NP_758860.1, NM 172353.2, NM_172359.2, NM_172361.2, NP 002380.3, NP722548.1, NP 758860.1, NP 758861.1, NP_758862.1, NP_758863.1,NP_758869.1, and NP_758871.1.

Useful genomic, polynucleotide and polypeptide information about humanCD55 (also known as complement decay-accelerating factor) are providedin, for example, the GeneCard Identifier GC01P207321, HGNC No. 2665,NCBI Gene ID 1604, Uniprot No. P08174, and NCBI RefSeq Nos. NM_000574.4,NM_001114752.2, NM_001300903.1, NM_001300904.1, NP_000565.1,NP_001108224.1, NP_001287832.1, and NP_001287833.1.

Useful genomic, polynucleotide and polypeptide information about humanCD59 are provided in, for example, the GeneCard Identifier GC11M033704,HGNC No. 1689, NCBI Gene ID 966, Uniprot No. P13987, and NCBI RefSeqNos. NP_000602.1, NM_000611.5, NP_001120695.1, NM_001127223.1, NP001120697.1, NM 001127225.1, NP_001120698.1, NM 001127226.1, NP001120699.1, NM 001127227.1, NP_976074.1, NM_203329.2, NP_976075.1, NM203330.2, NP 976076.1, and NM_203331.2.

Useful genomic, polynucleotide and polypeptide information about humanCD200 are provided in, for example, the GeneCard Identifier GC03P112332,HGNC No. 7203, NCBI Gene ID 4345, Uniprot No. P41217, and NCBI RefSeqNos. NP_001004196.2, NM_001004196.3, NP 001305757.1, NM 001318828.1,NP_005935.4, NM 005944.6, XP_005247539.1, and XM_005247482.2.

Useful genomic, polynucleotide and polypeptide information about humanHLA-C are provided in, for example, the GeneCard Identifier GC06M031272,HGNC No. 4933, NCBI Gene ID 3107, Uniprot No. P10321, and NCBI RefSeqNos. NP_002108.4 and NM_002117.5.

Useful genomic, polynucleotide and polypeptide information about humanHLA-E are provided in, for example, the GeneCard Identifier GC06P047281,HGNC No. 4962, NCBI Gene ID 3133, Uniprot No. P13747, and NCBI RefSeqNos. NP_005507.3 and NM_005516.5.

Useful genomic, polynucleotide and polypeptide information about humanHLA-G are provided in, for example, the GeneCard Identifier GC06P047256,HGNC No. 4964, NCBI Gene ID 3135, Uniprot No. P17693, and NCBI RefSeqNos. NP_002118.1 and NM_002127.5.

Useful genomic, polynucleotide and polypeptide information about humanPD-L1 or CD274 are provided in, for example, the GeneCard IdentifierGC09P005450, HGNC No. 17635, NCBI Gene ID 29126, Uniprot No. Q9NZQ7, andNCBI RefSeq Nos. NP_001254635.1, NM_001267706.1, NP_054862.1, andNM_014143.3.

Useful genomic, polynucleotide and polypeptide information about humanIDO1 are provided in, for example, the GeneCard Identifier GC08P039891,HGNC No. 6059, NCBI Gene ID 3620, Uniprot No. P14902, and NCBI RefSeqNos. NP_002155.1 and NM_002164.5.

Useful genomic, polynucleotide and polypeptide information about humanIL-10 are provided in, for example, the GeneCard Identifier GC01M206767,HGNC No. 5962, NCBI Gene ID 3586, Uniprot No. P22301, and NCBI RefSeqNos. NP_000563.1 and NM_000572.2.

Useful genomic, polynucleotide and polypeptide information about humanFas ligand (which is known as FasL, FASLG, CD178, TNFSF6, and the like)are provided in, for example, the GeneCard Identifier GC01P172628, HGNCNo. 11936, NCBI Gene ID 356, Uniprot No. P48023, and NCBI RefSeq Nos.NP_000630.1, NM_000639.2, NP_001289675.1, and NM_001302746.1.

Useful genomic, polynucleotide and polypeptide information about humanCCL21 are provided in, for example, the GeneCard Identifier GC09M034709,HGNC No. 10620, NCBI Gene ID 6366, Uniprot No. 000585, and NCBI RefSeqNos. NP_002980.1 and NM_002989.3.

Useful genomic, polynucleotide and polypeptide information about humanCCL22 are provided in, for example, the GeneCard Identifier GC16P057359,HGNC No. 10621, NCBI Gene ID 6367, Uniprot No. 000626, and NCBI RefSeqNos. NP_002981.2, NM_002990.4, XP_016879020.1, and XM_017023531.1.

Useful genomic, polynucleotide and polypeptide information about humanMfge8 are provided in, for example, the GeneCard Identifier GC15M088898,HGNC No. 7036, NCBI Gene ID 4240, Uniprot No. Q08431, and NCBI RefSeqNos. NP_001108086.1, NM_001114614.2, NP 001297248.1, NM 001310319.1, NP001297249.1, NM 001310320.1, NP_001297250.1, NM_001310321.1,NP_005919.2, and NM_005928.3.

Useful genomic, polynucleotide and polypeptide information about humanSerpinB9 are provided in, for example, the GeneCard IdentifierGC06M002887, HGNC No. 8955, NCBI Gene ID 5272, Uniprot No. P50453, andNCBI RefSeq Nos. NP_004146.1, NM_004155.5, XP_005249241.1, andXM_005249184.4.

Methods for modulating expression of genes and factors (proteins)include genome editing technologies, RNA or protein expressiontechnologies, and the like. For all of these technologies, well knownrecombinant techniques are used, to generate recombinant nucleic acidsas outlined herein.

In some embodiments, the cells (e.g., stem cell, induced pluripotentstem cell, differentiated cell, hematopoietic stem cell, primary T cellor CAR-T cell) possess genetic modifications that inactivate the B2M andCIITA genes and express a plurality of exogenous polypeptides selectedfrom the group including CD47 and DUX4, CD47 and CD24, CD47 and CD27,CD47 and CD46, CD47 and CD55, CD47 and CD59, CD47 and CD200, CD47 andHLA-C, CD47 and HLA-E, CD47 and HLA-E heavy chain, CD47 and HLA-G, CD47and PD-L1, CD47 and IDO1, CD47 and CTLA4-Ig, CD47 and C1-Inhibitor, CD47and IL-10, CD47 and IL-35, CD47 and TL-39, CD47 and FasL, CD47 andCCL21, CD47 and CCL22, CD47 and Mfge8, and CD47 and Serpinb9, and anycombination thereof. In some instances, such cells also possess agenetic modification that inactivates the CD142 gene.

In some instances, a gene editing system such as the CRISPR/Cas systemis used to facilitate the insertion of tolerogenic factors, such as thetolerogenic factors into a safe harbor or target locus, such as theAAVS1 locus, to actively inhibit immune rejection. In some instances,the tolerogenic factors are inserted into a safe harbor or target locususing an expression vector. In some embodiments, the safe harbor ortarget locus is an AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known asCD142), MICA, MICB, LRP1 (also known as CD91), HMGB1, ABO, RHD, FUT1, orKDM5D gene locus.

In some embodiments, expression of a target gene (e.g., DUX4, CD47, oranother tolerogenic factor gene) is increased by expression of fusionprotein or a protein complex containing (1) a site-specific bindingdomain specific for the endogenous target gene (e.g., DUX4, CD47, oranother tolerogenic factor gene) and (2) a transcriptional activator.

In some embodiments, the regulatory factor is comprised of a sitespecific DNA-binding nucleic acid molecule, such as a guide RNA (gRNA).In some embodiments, the method is achieved by site specific DNA-bindingtargeted proteins, such as zinc finger proteins (ZFP) or fusion proteinscontaining ZFP, which are also known as zinc finger nucleases (ZFNs).

In some embodiments, the regulatory factor comprises a site-specificbinding domain, such as using a DNA binding protein or DNA-bindingnucleic acid, which specifically binds to or hybridizes to the gene at atargeted region. In some embodiments, the provided polynucleotides orpolypeptides are coupled to or complexed with a site-specific nuclease,such as a modified nuclease. For example, in some embodiments, theadministration is effected using a fusion comprising a DNA-targetingprotein of a modified nuclease, such as a meganuclease or an RNA-guidednuclease such as a clustered regularly interspersed short palindromicnucleic acid (CRISPR)-Cas system, such as CRISPR-Cas9 system. In someembodiments, the nuclease is modified to lack nuclease activity. In someembodiments, the modified nuclease is a catalytically dead dCas9.

In some embodiments, the site specific binding domain may be derivedfrom a nuclease. For example, the recognition sequences of homingendonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI,I-TevII and I-TevIII. See also U.S. Pat. Nos. 5,420,032; 6,833,252;Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.,(1989) Gene 82:115-118; Perler et al, (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al., (1996)J. Mol. Biol. 263:163-180; Argast et al, (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, theDNA-binding specificity of homing endonucleases and meganucleases can beengineered to bind non-natural target sites. See, for example, Chevalieret al, (2002) Molec. Cell 10:895-905; Epinat et al, (2003) Nucleic AcidsRes. 31:2952-2962; Ashworth et al, (2006) Nature 441:656-659; Paques etal, (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.2007/0117128.

Zinc finger, TALE, and CRISPR system binding domains can be “engineered”to bind to a predetermined nucleotide sequence, for example viaengineering (altering one or more amino acids) of the recognition helixregion of a naturally occurring zinc finger or TALE protein. EngineeredDNA binding proteins (zinc fingers or TALEs) are proteins that arenon-naturally occurring. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No.20110301073.

In some embodiments, the site-specific binding domain comprises one ormore zinc-finger proteins (ZFPs) or domains thereof that bind to DNA ina sequence-specific manner. A ZFP or domain thereof is a protein ordomain within a larger protein that binds DNA in a sequence-specificmanner through one or more zinc fingers, regions of amino acid sequencewithin the binding domain whose structure is stabilized throughcoordination of a zinc ion.

Among the ZFPs are artificial ZFP domains targeting specific DNAsequences, typically 9-18 nucleotides long, generated by assembly ofindividual fingers. ZFPs include those in which a single finger domainis approximately 30 amino acids in length and contains an alpha helixcontaining two invariant histidine residues coordinated through zincwith two cysteines of a single beta turn, and having two, three, four,five, or six fingers. Generally, sequence-specificity of a ZFP may bealtered by making amino acid substitutions at the four helix positions(—1, 2, 3 and 6) on a zinc finger recognition helix. Thus, in someembodiments, the ZFP or ZFP-containing molecule is non-naturallyoccurring, e.g., is engineered to bind to a target site of choice. See,for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo etal. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635;7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528;2005/0267061, all incorporated herein by reference in their entireties.

Many gene-specific engineered zinc fingers are available commercially.For example, Sangamo Biosciences (Richmond, Calif., USA) has developed aplatform (CompoZr) for zinc-finger construction in partnership withSigma-Aldrich (St. Louis, Mo., USA), allowing investigators to bypasszinc-finger construction and validation altogether, and providesspecifically targeted zinc fingers for thousands of proteins (Gaj etal., Trends in Biotechnology, 2013, 31(7), 397-405). In someembodiments, commercially available zinc fingers are used or are customdesigned.

In some embodiments, the site-specific binding domain comprises anaturally occurring or engineered (non-naturally occurring)transcription activator-like protein (TAL) DNA binding domain, such asin a transcription activator-like protein effector (TALE) protein, See,e.g., U.S. Patent Publication No. 20110301073, incorporated by referencein its entirety herein.

In some embodiments, the site-specific binding domain is derived fromthe CRISPR/Cas system. In general, “CRISPR system” refers collectivelyto transcripts and other elements involved in the expression of ordirecting the activity of CRISPR-associated (“Cas”) genes, includingsequences encoding a Cas gene, a tracr (trans-activating CRISPR)sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-matesequence (encompassing a “direct repeat” and a tracrRNA-processedpartial direct repeat in the context of an endogenous CRISPR system), aguide sequence (also referred to as a “spacer” in the context of anendogenous CRISPR system, or a “targeting sequence”), and/or othersequences and transcripts from a CRISPR locus.

In general, a guide sequence includes a targeting domain comprising apolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of the CRISPR complex to the target sequence.In some embodiments, the degree of complementarity between a guidesequence and its corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some examples, thetargeting domain of the gRNA is complementary, e.g., at least 80, 85,90, 95, 98 or 99% complementary, e.g., fully complementary, to thetarget sequence on the target nucleic acid.

In some embodiments, the target site is upstream of a transcriptioninitiation site of the target gene. In some embodiments, the target siteis adjacent to a transcription initiation site of the gene. In someembodiments, the target site is adjacent to an RNA polymerase pause sitedownstream of a transcription initiation site of the gene.

In some embodiments, the targeting domain is configured to target thepromoter region of the target gene to promote transcription initiation,binding of one or more transcription enhancers or activators, and/or RNApolymerase. One or more gRNA can be used to target the promoter regionof the gene. In some embodiments, one or more regions of the gene can betargeted. In certain aspects, the target sites are within 600 base pairson either side of a transcription start site (TSS) of the gene.

It is within the level of a skilled artisan to design or identify a gRNAsequence that is or comprises a sequence targeting a gene, including theexon sequence and sequences of regulatory regions, including promotersand activators. A genome-wide gRNA database for CRISPR genome editing ispublicly available, which contains exemplary single guide RNA (sgRNA)target sequences in constitutive exons of genes in the human genome ormouse genome (see e.g., genescript.com/gRNA-database.html; see also,Sanjana et al. (2014) Nat. Methods, 11:783-4; www.e-crisp.org/E-CRISP/;crispr.mit.edu/). In some embodiments, the gRNA sequence is or comprisesa sequence with minimal off-target binding to a non-target gene.

In some embodiments, the regulatory factor further comprises afunctional domain, e.g., a transcriptional activator.

In some embodiments, the transcriptional activator is or contains one ormore regulatory elements, such as one or more transcriptional controlelements of a target gene, whereby a site-specific domain as providedabove is recognized to drive expression of such gene. In someembodiments, the transcriptional activator drives expression of thetarget gene. In some cases, the transcriptional activator, can be orcontain all or a portion of an heterologous transactivation domain. Forexample, in some embodiments, the transcriptional activator is selectedfrom Herpes simplex-derived transactivation domain, Dnmt3amethyltransferase domain, p65, VP16, and VP64.

In some embodiments, the regulatory factor is a zinc fingertranscription factor (ZF-TF). In some embodiments, the regulatory factoris VP64-p65-Rta (VPR).

In certain embodiments, the regulatory factor further comprises atranscriptional regulatory domain. Common domains include, e.g.,transcription factor domains (activators, repressors, co-activators,co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max,mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymesand their associated factors and modifiers; DNA rearrangement enzymesand their associated factors and modifiers; chromatin associatedproteins and their modifiers (e.g., kinases, acetylases anddeacetylases); and DNA modifying enzymes (e.g., methyltransferases suchas members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L,etc., topoisomerases, helicases, ligases, kinases, phosphatases,polymerases, endonucleases) and their associated factors and modifiers.See, e.g., U.S. Publication No. 2013/0253040, incorporated by referencein its entirety herein.

Suitable domains for achieving activation include the HSV VP 16activation domain (see, e.g., Hagmann et al, J. Virol. 71, 5952-5962 (197)) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin.Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappaB (Bitko & Bank, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Beerliet al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron(Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplaryactivation domains include, Oct 1, Oct-2A, Spl, AP-2, and CTF1 (Seipelet al, EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. See, for example, Robyr et al, (2000) Mol.Endocrinol. 14:329-347; Collingwood et al, (1999) J. Mol. Endocrinol23:255-275; Leo et al, (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol. 46:77-89; McKenna et al, (1999) J. SteroidBiochem. Mol. Biol. 69:3-12; Malik et al, (2000) Trends Biochem. Sci.25:277-283; and Lemon et al, (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, AP1, ARF-5, -6,-1, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRAB1, See, for example, Ogawa et al, (2000) Gene 245:21-29; Okanamiet al, (1996) Genes Cells 1:87-99; Goff et al, (1991) Genes Dev.5:298-309; Cho et al, (1999) Plant Mol Biol 40:419-429; Ulmason et al,(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al,(2000) Plant J. 22:1-8; Gong et al, (1999) Plant Mol. Biol. 41:33-44;and Hobo et al., (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

Exemplary repression domains that can be used to make genetic repressorsinclude, but are not limited to, KRAB A/B, KOX, TGF-beta-inducible earlygene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g.,DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), Rb, and MeCP2. See, for example,Bird et al, (1999) Cell 99:451-454; Tyler et al, (1999) Cell 99:443-446;Knoepfler et al, (1999) Cell 99:447-450; and Robertson et al, (2000)Nature Genet. 25:338-342. Additional exemplary repression domainsinclude, but are not limited to, ROM2 and AtHD2A. See, for example, Chemet al, (1996) Plant Cell 8:305-321; and Wu et al, (2000) Plant J.22:19-27.

In some instances, the domain is involved in epigenetic regulation of achromosome. In some embodiments, the domain is a histoneacetyltransferase (HAT), e.g., type-A, nuclear localized such as MYSTfamily members MOZ, Ybf2/Sas3, MOF, and Tip60, GNAT family members Gcn5or pCAF, the p300 family members CBP, p300 or Rttl09 (Bemdsen and Denu(2008) Curr Opin Struct Biol 18(6):682-689). In other instances thedomain is a histone deacetylase (HD AC) such as the class I (HDAC-1, 2,3, and 8), class II (HDAC IIA (HDAC-4, 5, 7 and 9), HD AC IIB (HDAC 6and 10)), class IV (HDAC-l 1), class III (also known as sirtuins(SIRTs); SIRT1-7) (see Mottamal et al., (2015) Molecules20(3):3898-3941). Another domain that is used in some embodiments is ahistone phosphorylase or kinase, where examples include MSK1, MSK2, ATR,ATM, DNA-PK, Bubl, VprBP, IKK-a, PKCpi, Dik/Zip, JAK2, PKC5, WSTF andCK2. In some embodiments, a methylation domain is used and may be chosenfrom groups such as Ezh2, PRMT1/6, PRMT5/7, PRMT 2/6, CARM1, set7/9,MLL, ALL-1, Suv 39h, G9a, SETDB1, Ezh2, Set2, Dotl, PRMT 1/6, PRMT 5/7,PR-Set7 and Suv4-20h, Domains involved in sumoylation and biotinylation(Lys9, 13, 4, 18 and 12) may also be used in some embodiments (reviewsee Kousarides (2007) Cell 128:693-705).

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well known to those of skill in the art. Fusionmolecules comprise a DNA-binding domain and a functional domain (e.g., atranscriptional activation or repression domain). Fusion molecules alsooptionally comprise nuclear localization signals (such as, for example,that from the SV40 medium T-antigen) and epitope tags (such as, forexample, FLAG and hemagglutinin). Fusion proteins (and nucleic acidsencoding them) are designed such that the translational reading frame ispreserved among the components of the fusion.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp et al, (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935. Likewise, CRISPR/Cas TFs and nucleases comprisinga sgRNA nucleic acid component in association with a polypeptidecomponent function domain are also known to those of skill in the artand detailed herein.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express CD47. In some embodiments, the presentdisclosure provides a method for altering a cell genome to express CD47.In certain embodiments, at least one ribonucleic acid or at least onepair of ribonucleic acids may be utilized to facilitate the insertion ofCD47 into a cell line. In certain embodiments, the at least oneribonucleic acid or the at least one pair of ribonucleic acids isselected from the group consisting of SEQ ID NOS:200784-231885 of Table29 of WO2016183041, which is herein incorporated by reference.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express HLA-C. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressHLA-C. In certain embodiments, at least one ribonucleic acid or at leastone pair of ribonucleic acids may be utilized to facilitate theinsertion of HLA-C into a cell line. In certain embodiments, the atleast one ribonucleic acid or the at least one pair of ribonucleic acidsis selected from the group consisting of SEQ ID NOS:3278-5183 of Table10 of WO2016183041, which is herein incorporated by reference.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express HLA-E. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressHLA-E. In certain embodiments, at least one ribonucleic acid or at leastone pair of ribonucleic acids may be utilized to facilitate theinsertion of HLA-E into a cell line. In certain embodiments, the atleast one ribonucleic acid or the at least one pair of ribonucleic acidsis selected from the group consisting of SEQ ID NOS:189859-193183 ofTable 19 of WO2016183041, which is herein incorporated by reference.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express HLA-F. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressHLA-F. In certain embodiments, at least one ribonucleic acid or at leastone pair of ribonucleic acids may be utilized to facilitate theinsertion of HLA-F into a cell line. In certain embodiments, the atleast one ribonucleic acid or the at least one pair of ribonucleic acidsis selected from the group consisting of SEQ ID NOS: 688808-399754 ofTable 45 of WO2016183041, which is herein incorporated by reference.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express HLA-G. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressHLA-G. In certain embodiments, at least one ribonucleic acid or at leastone pair of ribonucleic acids may be utilized to facilitate theinsertion of HLA-G into a stem cell line. In certain embodiments, the atleast one ribonucleic acid or the at least one pair of ribonucleic acidsis selected from the group consisting of SEQ ID NOS:188372-189858 ofTable 18 of WO2016183041, which is herein incorporated by reference.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express PD-L1. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressPD-L1. In certain embodiments, at least one ribonucleic acid or at leastone pair of ribonucleic acids may be utilized to facilitate theinsertion of PD-L1 into a stem cell line. In certain embodiments, the atleast one ribonucleic acid or the at least one pair of ribonucleic acidsis selected from the group consisting of SEQ ID NOS:193184-200783 ofTable 21 of WO2016183041, which is herein incorporated by reference.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express CTLA4-Ig. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressCTLA4-Ig. In certain embodiments, at least one ribonucleic acid or atleast one pair of ribonucleic acids may be utilized to facilitate theinsertion of CTLA4-Ig into a stem cell line. In certain embodiments, theat least one ribonucleic acid or the at least one pair of ribonucleicacids is selected from any one disclosed in WO2016183041, including thesequence listing.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express CI-inhibitor. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressCI-inhibitor. In certain embodiments, at least one ribonucleic acid orat least one pair of ribonucleic acids may be utilized to facilitate theinsertion of CI-inhibitor into a stem cell line. In certain embodiments,the at least one ribonucleic acid or the at least one pair ofribonucleic acids is selected from any one disclosed in WO2016183041,including the sequence listing.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express IL-35. In some embodiments, the presentdisclosure provides a method for altering a cell genome to expressIL-35. In certain embodiments, at least one ribonucleic acid or at leastone pair of ribonucleic acids may be utilized to facilitate theinsertion of IL-35 into a stem cell line. In certain embodiments, the atleast one ribonucleic acid or the at least one pair of ribonucleic acidsis selected from any one disclosed in WO2016183041, including thesequence listing.

In some embodiments, the tolerogenic factors are expressed in a cellusing an expression vector. In some embodiments, the tolerogenic factorsare introduced to the cell using a viral expression vector that mediatesintegration of the tolerogenic factor sequence into the genome of thecell. For example, the expression vector for expressing CD47 in a cellcomprises a polynucleotide sequence encoding CD47. The expression vectorcan be an inducible expression vector. The expression vector can be aviral vector, such as but not limited to, a lentiviral vector. In someembodiments, the tolerogenic factors are introduced into the cells usingfusogen-mediated delivery or a transposase system selected from thegroup consisting of conditional or inducible transposases, conditionalor inducible PiggyBac transposons, conditional or inducible SleepingBeauty (SB11) transposons, conditional or inducible Mos1 transposons,and conditional or inducible Tol2 transposons.

In some embodiments, the present disclosure provides a cell (e.g., aprimary T cell and a hypoimmunogenic stem cell and derivative thereof)or population thereof comprising a genome in which the cell genome hasbeen modified to express any one of the polypeptides selected from thegroup consisting of HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5,B2M, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR,4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS,LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3,VISTA, TLT, PD-L2, CD58, CD2, HELIOS, and IDO1. In some embodiments, thepresent disclosure provides a method for altering a cell genome toexpress any one of the polypeptides selected from the group consistingof HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5, B2M, RFX5, RFX-AP,HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR, 4-1BB, CD28, B7-1,CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3,CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2,CD58, CD2, HELIOS, and IDOL. In certain embodiments, at least oneribonucleic acid or at least one pair of ribonucleic acids may beutilized to facilitate the insertion of the selected polypeptide into astem cell line. In certain embodiments, the at least one ribonucleicacid or the at least one pair of ribonucleic acids is selected from anyone disclosed in Appendices 1-47 and the sequence listing ofWO2016183041, the disclosure is incorporated herein by references.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cassystem or any of the gene editing systems described herein) is used tofacilitate the insertion of a polynucleotide encoding a tolerogenicfactor, into a genomic locus of the hypoimmunogenic cell. In some cases,the polynucleotide encoding the tolerogenic factor is inserted into asafe harbor or target locus, such as but not limited to, an AAVS1, CCR5,CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO,RHD, FUT1, or KDM5D gene locus. In some embodiments, the polynucleotideencoding the tolerogenic factor is inserted into a B2M gene locus, aCIITA gene locus, a TRAC gene locus, or a TRB gene locus. In someembodiments, the polynucleotide encoding the tolerogenic factor isinserted into any one of the gene loci depicted in Table 15 providedherein. In certain embodiments, the polynucleotide encoding thetolerogenic factor is operably linked to a promoter.

In some embodiments, the cells are engineered to expresses an increasedamount of one or more of CD47, DUX4, CD24, CD27, CD35, CD46, CD55, CD59,CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig,C1-Inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, CD16, CD52,H2-M3, CD16 Fc receptor, IL15-RF, and/or Serpinb9 relative to a cell ofthe same cell type that does not comprise the modifications.

N. Chimeric Antigen Receptors

Provided herein are hypoimmunogenic cells comprising a chimeric antigenreceptor (CAR). In some embodiments, the CAR binds to CD19. In someembodiments, the CAR binds to CD22. In some embodiments, the CAR bindsto CD19. In some embodiments, the CAR binds to CD19 and CD22. In someembodiments, the CAR is selected from the group consisting of a firstgeneration CAR, a second generation CAR, a third generation CAR, and afourth generation CAR. In some embodiments, the CAR includes a singlebinding domain that binds to a single target antigen. In someembodiments, the CAR includes a single binding domain that binds to morethan one target antigen, e.g., 2, 3, or more target antigens. In someembodiments, the CAR includes two binding domains such that each bindingdomain binds to a different target antigens. In some embodiments, theCAR includes two binding domains such that each binding domain binds tothe same target antigen. Detailed descriptions of exemplary CARsincluding CD19-specific, CD22-specific and CD19/CD22-bispecific CARs canbe found in WO2012/079000, WO2016/149578 and WO2020/014482, thedisclosures including the sequence listings and figures are incorporatedherein by reference in their entirety.

In some embodiments, the CD19 specific CAR includes an anti-CD19single-chain antibody fragment (scFv), a transmembrane domain such asone derived from human CD8α, a 4-1BB (CD137) co-stimulatory signalingdomain, and a CD3ζ signaling domain. In some embodiments, the CD22specific CAR includes an anti-CD22 scFv, a transmembrane domain such asone derived from human CD8α, a 4-1BB (CD137) co-stimulatory signalingdomain, and a CD3ζ signaling domain. In some embodiments, theCD19/CD22-bispecific CAR includes an anti-CD19 scFv, an anti-CD22 scFv,a transmembrane domain such as one derived from human CD8α, a 4-1BB(CD137) co-stimulatory signaling domain, and a CD3ζ signaling domain.

In some embodiments, the CAR comprises a commercial CAR constructcarried by a T cell. Non-limiting examples of commercial CAR-T cellbased therapies include brexucabtagene autoleucel (TECARTUS®),axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®),lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®),Descartes-08 and Descartes-11 from Cartesian Therapeutics, CTL110 fromNovartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from AutolusLimited, UCARTCS from Cellectis, PBCAR19B and PBCAR269A from PrecisionBiosciences, FT819 from Fate Therapeutics, and CYAD-211 from ClyadOncology.

In some embodiments, a hypoimmunogenic cell described herein comprises apolynucleotide encoding a chimeric antigen receptor (CAR) comprising anantigen binding domain. In some embodiments, a hypoimmunogenic celldescribed herein comprises a chimeric antigen receptor (CAR) comprisingan antigen binding domain. In some embodiments, the polynucleotide is orcomprises a chimeric antigen receptor (CAR) comprising an antigenbinding domain. In some embodiments, the CAR is or comprises a firstgeneration CAR comprising an antigen binding domain, a transmembranedomain, and at least one signaling domain (e.g., one, two or threesignaling domains). In some embodiments, the CAR comprises a secondgeneration CAR comprising an antigen binding domain, a transmembranedomain, and at least two signaling domains. In some embodiments, the CARcomprises a third generation CAR comprising an antigen binding domain, atransmembrane domain, and at least three signaling domains. In someembodiments, a fourth generation CAR comprising an antigen bindingdomain, a transmembrane domain, three or four signaling domains, and adomain which upon successful signaling of the CAR induces expression ofa cytokine gene. In some embodiments, the antigen binding domain is orcomprises an antibody, an antibody fragment, an scFv or a Fab.

1. Antigen Binding Domain (ABD) Targets an Antigen Characteristic of aNeoplastic or Cancer Cell

In some embodiments, the antigen binding domain (ABD) targets an antigencharacteristic of a neoplastic cell. In other words, the antigen bindingdomain targets an antigen expressed by a neoplastic or cancer cell. Insome embodiments, the ABD binds a tumor associated antigen. In someembodiments, the antigen characteristic of a neoplastic cell (e.g.,antigen associated with a neoplastic or cancer cell) or a tumorassociated antigen is selected from a cell surface receptor, an ionchannel-linked receptor, an enzyme-linked receptor, a G protein-coupledreceptor, receptor tyrosine kinase, tyrosine kinase associated receptor,receptor-like tyrosine phosphatase, receptor serine/threonine kinase,receptor guanylyl cyclase, histidine kinase associated receptor,epidermal growth factor receptors (EGFR) (including ErbB1/EGFR,ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), fibroblast growth factorreceptors (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7,FGF18, and FGF21), vascular endothelial growth factor receptors (VEGFR)(including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF), RET Receptor andthe Eph Receptor Family (including EphA1, EphA2, EphA3, EphA4, EphA5,EphA6, EphA7, EphA8, EphA9, EphA10, EphB1, EphB2. EphB3, EphB4, andEphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb,Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters,NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9,sphingosin-1-phosphate receptor (S1P1R), NMDA channel, transmembraneprotein, multispan transmembrane protein, T-cell receptor motifs, T-cellalpha chains, T-cell β chains, T-cell γ chains, T-cell δ chains, CCR7,CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22,CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68,CD80, CD95, CD117, CD127, CD133, CD137 (4-1BB), CD163, F4/80, IL-4Ra,Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor,NKp46, perforin, CD4+, Th1, Th2, Th17, Th40, Th22, Th9, Tfh, canonicalTreg. FoxP3+, Tr1, Th3, Treg17, T_(RE)G; CDCP, NT5E, EpCAM, CEA, gpA33,mucins, TAG-72, carbonic anhydrase IX, PSMA, folate binding protein,gangliosides (e.g., CD2, CD3, GM2), Lewis-γ², VEGF, VEGFR 1/2/3, αVβ3,α5β1, ErbB1/EGFR, ErbB1/HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1,TRAIL-R2, RANKL, FAP, Tenascin, PDL-1, BAFF, HDAC, ABL, FLT3, KIT, MET,RET, IL-1β, ALK, RANKL, mTOR, CTLA-4, IL-6, IL-6R, JAK3, BRAF, PTCH,Smoothened, PIGF, ANPEP, TIMP1, PLAUR, PTPRJ, LTBR, ANTXR1, folatereceptor alpha (FRa), ERBB2 (Her2/neu), EphA2, IL-13Ra2, epidermalgrowth factor receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22,CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, MUC16 (CA125),L1CAM, LeY, MSLN, IL13Rα1, L1-CAM, Tn Ag, prostate specific membraneantigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3,KIT, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY,CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4,CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-1 receptor, CAIX,LMP2, gp100, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA,o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D,CXORF61, CD97, CD179a, ALK, Polysialic acid, PLACl, GloboH, NY-BR-1,UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1,LAGE-la, MAGE-A1, legumain, HPV E6, E7, ETV6-AML, sperm protein 17,XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, major histocompatibility complex classI-related gene protein (MR1), urokinase-type plasminogen activatorreceptor (uPAR), Fos-related antigen 1, p53, p53 mutant, prostein,survivin, telomerase, PCTA-1/Galectin 8, MelanA/MARTI, Ras mutant,hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETSfusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC,TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1,human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF,CLECi2A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, a neoantigen, CD133,CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C,(HLA-A,B,C) CD49f, CD151 CD340, CD200, tkrA, trkB, or trkC, or anantigenic fragment or antigenic portion thereof.

2. ABD Targets an Antigen Characteristic of a T Cell

In some embodiments, the antigen binding domain targets an antigencharacteristic of a T cell. In some embodiments, the ABD binds anantigen associated with a T cell. In some instances, such an antigen isexpressed by a T cell or is located on the surface of a T cell. In someembodiments, the antigen characteristic of a T cell or the T cellassociated antigen is selected from a cell surface receptor, a membranetransport protein (e.g., an active or passive transport protein such as,for example, an ion channel protein, a pore-forming protein, etc.), atransmembrane receptor, a membrane enzyme, and/or a cell adhesionprotein characteristic of a T cell. In some embodiments, an antigencharacteristic of a T cell may be a G protein-coupled receptor, receptortyrosine kinase, tyrosine kinase associated receptor, receptor-liketyrosine phosphatase, receptor serine/threonine kinase, receptorguanylyl cyclase, histidine kinase associated receptor, AKT1; AKT2;AKT3; ATF2; BCL10; CALM1; CD3D (CD36); CD3E (CD3E); CD3G (CD37); CD4;CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3ζ); CTLA-4 (CD152);ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; HLA-DRA;HLA-DRB1; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS; IKBKA (CHUK); IKBKB;IKBKE; IKBKG (NEMO); IL2; ITPR1; ITK; JUN; KRAS2; LAT; LCK; MAP2K1(MEK1); MAP2K2 (MEK2); MAP2K3 (MKK3); MAP2K4 (MKK4); MAP2K6 (MKK6);MAP2K7 (MKK7); MAP3K1 (MEKK1); MAP3K3; MAP3K4; MAP3K5; MAP3K8; MAP3K14(NIK); MAPK8 (JNK1); MAPK9 (JNK2); MAPK10 (JNK3); MAPK11 (p380); MAPK12(p387); MAPK13 (p386); MAPK14 (p38a); NCK; NFAT1; NFAT2; NFKB1; NFKB2;NFKBIA; NRAS; PAK1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA;PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (Perforin);PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC;TRAF6; VAV1; VAV2; or ZAP70.

3. ABD Targets an Antigen Characteristic of an Autoimmune orInflammatory Disorder

In some embodiments, the antigen binding domain targets an antigencharacteristic of an autoimmune or inflammatory disorder. In someembodiments, the ABD binds an antigen associated with an autoimmune orinflammatory disorder. In some instances, the antigen is expressed by acell associated with an autoimmune or inflammatory disorder. In someembodiments, the autoimmune or inflammatory disorder is selected fromchronic graft-vs-host disease (GVHD), lupus, arthritis, immune complexglomerulonephritis, goodpasture syndrome, uveitis, hepatitis, systemicsclerosis or scleroderma, type I diabetes, multiple sclerosis, coldagglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmunehemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thromboticthrombocytopenia purrpura, neuromyelits optica, Evan's syndrome, IgMmediated neuropathy, cryoglobulinemia, dermatomyositis, idiopathicthrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquiredangioedema, chronic urticarial, antiphospholipid demyelinatingpolyneuropathy, and autoimmune thrombocytopenia or neutropenia or purered cell aplasias, while exemplary non-limiting examples of alloimmunediseases include allosensitization (see, for example, Blazar et al.,2015, Am. J. Transplant, 15(4):931-41) or xenosensitization fromhematopoietic or solid organ transplantation, blood transfusions,pregnancy with fetal allosensitization, neonatal alloimmunethrombocytopenia, hemolytic disease of the newborn, sensitization toforeign antigens such as can occur with replacement of inherited oracquired deficiency disorders treated with enzyme or protein replacementtherapy, blood products, and gene therapy. In some embodiments, theantigen characteristic of an autoimmune or inflammatory disorder isselected from a cell surface receptor, an ion channel-linked receptor,an enzyme-linked receptor, a G protein-coupled receptor, receptortyrosine kinase, tyrosine kinase associated receptor, receptor-liketyrosine phosphatase, receptor serine/threonine kinase, receptorguanylyl cyclase, or histidine kinase associated receptor.

In some embodiments, an antigen binding domain of a CAR binds to aligand expressed on B cells, plasma cells, or plasmablasts. In someembodiments, an antigen binding domain of a CAR binds to CD10, CD19,CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF,interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2. See,e.g., US 2003/0077249; WO 2017/058753; WO 2017/058850, the contents ofwhich are herein incorporated by reference.

4. ABD Targets an Antigen Characteristic of Senescent Cells

In some embodiments, the antigen binding domain targets an antigencharacteristic of senescent cells, e.g., urokinase-type plasminogenactivator receptor (uPAR). In some embodiments, the ABD binds an antigenassociated with a senescent cell. In some instances, the antigen isexpressed by a senescent cell. In some embodiments, the CAR may be usedfor treatment or prophylaxis of disorders characterized by the aberrantaccumulation of senescent cells, e.g., liver and lung fibrosis,atherosclerosis, diabetes and osteoarthritis.

5. ABD Targets an Antigen Characteristic of an Infectious Disease

In some embodiments, the antigen binding domain targets an antigencharacteristic of an infectious disease. In some embodiments, the ABDbinds an antigen associated with an infectious disease. In someinstances, the antigen is expressed by a cell affected by an infectiousdisease. In some embodiments, wherein the infectious disease is selectedfrom HIV, hepatitis B virus, hepatitis C virus, Human herpes virus,Human herpes virus 8 (HHV-8, Kaposi sarcoma-associated herpes virus(KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cellpolyomavirus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV,human papillomavirus. In some embodiments, the antigen characteristic ofan infectious disease is selected from a cell surface receptor, an ionchannel-linked receptor, an enzyme-linked receptor, a G protein-coupledreceptor, receptor tyrosine kinase, tyrosine kinase associated receptor,receptor-like tyrosine phosphatase, receptor serine/threonine kinase,receptor guanylyl cyclase, histidine kinase associated receptor, HIVEnv, gpl20, or CD4-induced epitope on HIV-1 Env.

6. ABD Binds to a Cell Surface Antigen of a Cell

In some embodiments, an antigen binding domain binds to a cell surfaceantigen of a cell. In some embodiments, a cell surface antigen ischaracteristic of (e.g., expressed by) a particular or specific celltype. In some embodiments, a cell surface antigen is characteristic ofmore than one type of cell.

In some embodiments, a CAR antigen binding domain binds a cell surfaceantigen characteristic of a T cell, such as a cell surface antigen on aT cell. In some embodiments, an antigen characteristic of a T cell maybe a cell surface receptor, a membrane transport protein (e.g., anactive or passive transport protein such as, for example, an ion channelprotein, a pore-forming protein, etc.), a transmembrane receptor, amembrane enzyme, and/or a cell adhesion protein characteristic of a Tcell. In some embodiments, an antigen characteristic of a T cell may bea G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinaseassociated receptor, receptor-like tyrosine phosphatase, receptorserine/threonine kinase, receptor guanylyl cyclase, or histidine kinaseassociated receptor.

In some embodiments, an antigen binding domain of a CAR binds a T cellreceptor. In some embodiments, a T cell receptor may be AKT1; AKT2;AKT3; ATF2; BCL10; CALM1; CD3D (CD36); CD3E (CD3R); CD3G (CD37); CD4;CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3ζ); CTLA-4 (CD152);ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; HLA-DRA;HLA-DRB1; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS; IKBKA (CHUK); IKBKB;IKBKE; IKBKG (NEMO); IL2; ITPR1; ITK; JUN; KRAS2; LAT; LCK; MAP2K1(MEK1); MAP2K2 (MEK2); MAP2K3 (MKK3); MAP2K4 (MKK4); MAP2K6 (MKK6);MAP2K7 (MKK7); MAP3K1 (MEKK1); MAP3K3; MAP3K4; MAP3K5; MAP3K8; MAP3K14(NIK); MAPK8 (JNK1); MAPK9 (JNK2); MAPK10 (JNK3); MAPK11 (p380); MAPK12(p387); MAPK13 (p386); MAPK14 (p38a); NCK; NFAT1; NFAT2; NFKB1; NFKB2;NFKBIA; NRAS; PAK1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA;PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (Perforin);PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC;TRAF6; VAV1; VAV2; or ZAP70.

7. Transmembrane Domain

In some embodiments, the CAR transmembrane domain comprises at least atransmembrane region of the alpha, beta or zeta chain of a T cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variantthereof. In some embodiments, the transmembrane domain comprises atleast a transmembrane region(s) of CD8α, CD8β, 4-1BB/CD137, CD28, CD34,CD4, FcεRIγ, CD16, OX40/CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ,CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40,CD40L/CD154, VEGFR2, FAS, and FGFR2B, or functional variant thereofantigen binding domain binds

8. Signaling Domain or Plurality of Signaling Domains

In some embodiments, a CAR described herein comprises one or at leastone signaling domain selected from one or more of B7-1/CD80; B7-2/CD86;B7-H1/PD-L1; B7-H2; B7-H3; B7-H4; B7-H6; B7-H7; BTLA/CD272; CD28;CTLA-4; Gi24/VISTA/B7-H5; ICOS/CD278; PD-1; PD-L2/B7-DC; PDCD6);4-1BB/TNFSF9/CD137; 4-1n Ligand/TNFSF9; BAFF/BLyS/TNFSF13B; BAFFR/TNFRSF13C; CD27/TNFRSF7; CD27 Ligand/TNFSF7; CD30/TNFRSF8; CD30Ligand/TNFSF8; CD40/TNFRSF5; CD40/TNFSF5; CD40 Ligand/TNFSF5;DR3/TNFRSF25; GITR/TNFRSF18; GITR Ligand/TNFSF18; HVEM/TNFRSF14;LIGHT/TNFSF14; Lymphotoxin-alpha/TNF-beta; OX40/TNFRSF4; OX40Ligand/TNFSF4; RELT/TNFRSF19L; TACI/TNFRSF13B; TL1A/TNFSF15; TNF-alpha;TNF RII/TNFRSF1B); 2B4/CD244/SLAMF4; BLAME/SLAMF8; CD2; CD2F-10/SLAMF9;CD48/SLAMF2; CD58/LFA-3; CD84/SLAMF5; CD229/SLAMF3; CRACC/SLAMF7;NTB-A/SLAMF6; SLAM/CD150); CD2; CD7; CD53; CD82/Kai-1; CD90/Thy1; CD96;CD160; CD200; CD300a/LMIR1; HLA Class I; HLA-DR; Ikaros; Integrin alpha4/CD49d; Integrin alpha 4 beta 1; Integrin alpha 4 beta 7/LPAM-1; LAG-3;TCL1A; TCL1B; CRTAM; DAP12; Dectin-1/CLEC7A; DPPIV/CD26; EphB6;TIM-1/KIM-1/HAVCR; TIM-4; TSLP; TSLP R; lymphocyte function associatedantigen-1 (LFA-1); NKG2C, a CD3 zeta domain, an immunoreceptortyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134/OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically bindswith CD83, or functional fragment thereof.

In some embodiments, the at least one signaling domain comprises a CD3zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM),or functional variant thereof. In other embodiments, the at least onesignaling domain comprises (i) a CD3 zeta domain, or an immunoreceptortyrosine-based activation motif (ITAM), or functional variant thereof;and (ii) a CD28 domain, or a 4-1BB domain, or functional variantthereof. In yet other embodiments, the at least one signaling domaincomprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-basedactivation motif (ITAM), or functional variant thereof, (ii) a CD28domain or functional variant thereof, and (iii) a 4-1BB domain, or aCD134 domain, or functional variant thereof. In some embodiments, the atleast one signaling domain comprises a (i) a CD3 zeta domain, or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof; (ii) a CD28 domain or functional variant thereof, (iii)a 4-1BB domain, or a CD134 domain, or functional variant thereof; and(iv) a cytokine or costimulatory ligand transgene.

In some embodiments, the at least two signaling domains comprise a CD3zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM),or functional variant thereof. In other embodiments, the at least twosignaling domains comprise (i) a CD3 zeta domain, or an immunoreceptortyrosine-based activation motif (ITAM), or functional variant thereof;and (ii) a CD28 domain, or a 4-1BB domain, or functional variantthereof. In yet other embodiments, the at least one signaling domaincomprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-basedactivation motif (ITAM), or functional variant thereof, (ii) a CD28domain or functional variant thereof, and (iii) a 4-1BB domain, or aCD134 domain, or functional variant thereof. In some embodiments, the atleast two signaling domains comprise a (i) a CD3 zeta domain, or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof; (ii) a CD28 domain or functional variant thereof, (iii)a 4-1BB domain, or a CD134 domain, or functional variant thereof; and(iv) a cytokine or costimulatory ligand transgene.

In some embodiments, the at least three signaling domains comprise a CD3zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM),or functional variant thereof. In other embodiments, the at least threesignaling domains comprise (i) a CD3 zeta domain, or an immunoreceptortyrosine-based activation motif (ITAM), or functional variant thereof;and (ii) a CD28 domain, or a 4-1BB domain, or functional variantthereof. In yet other embodiments, the least three signaling domainscomprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-basedactivation motif (ITAM), or functional variant thereof; (ii) a CD28domain or functional variant thereof, and (iii) a 4-1BB domain, or aCD134 domain, or functional variant thereof. In some embodiments, the atleast three signaling domains comprise a (i) a CD3 zeta domain, or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof, (ii) a CD28 domain or functional variant thereof; (iii)a 4-1BB domain, or a CD134 domain, or functional variant thereof, and(iv) a cytokine or costimulatory ligand transgene.

In some embodiments, the CAR comprises a CD3 zeta domain or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof. In some embodiments, the CAR comprises (i) a CD3 zetadomain, or an immunoreceptor tyrosine-based activation motif (ITAM), orfunctional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain,or functional variant thereof.

In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof; (ii) a CD28 domain or functional variant thereof, and(iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.

In some embodiments, the CAR comprises (i) a CD3 zeta domain, or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functionalvariant thereof, and/or (iii) a 4-1BB domain, or a CD134 domain, orfunctional variant thereof.

In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or animmunoreceptor tyrosine-based activation motif (ITAM), or functionalvariant thereof; (ii) a CD28 domain or functional variant thereof, (iii)a 4-1BB domain, or a CD134 domain, or functional variant thereof; and(iv) a cytokine or costimulatory ligand transgene.

9. Domain which Upon Successful Signaling of the CAR Induces Expressionof a Cytokine Gene

In some embodiments, a first, second, third, or fourth generation CARfurther comprises a domain which upon successful signaling of the CARinduces expression of a cytokine gene. In some embodiments, a cytokinegene is endogenous or exogenous to a target cell comprising a CAR whichcomprises a domain which upon successful signaling of the CAR inducesexpression of a cytokine gene. In some embodiments, a cytokine geneencodes a pro-inflammatory cytokine. In some embodiments, a cytokinegene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, or IFN-gamma, orfunctional fragment thereof. In some embodiments, a domain which uponsuccessful signaling of the CAR induces expression of a cytokine gene isor comprises a transcription factor or functional domain or fragmentthereof. In some embodiments, a domain which upon successful signalingof the CAR induces expression of a cytokine gene is or comprises atranscription factor or functional domain or fragment thereof. In someembodiments, a transcription factor or functional domain or fragmentthereof is or comprises a nuclear factor of activated T cells (NFAT), anNF-kB, or functional domain or fragment thereof. See, e.g., Zhang. C. etal., Engineering CAR-T cells. Biomarker Research. 5:22 (2017); WO2016126608; Sha, H. et al. Chimaeric antigen receptor T-cell therapy fortumour immunotherapy. Bioscience Reports Jan. 27, 2017, 37 (1).

In some embodiments, the CAR further comprises one or more spacers,e.g., wherein the spacer is a first spacer between the antigen bindingdomain and the transmembrane domain. In some embodiments, the firstspacer includes at least a portion of an immunoglobulin constant regionor variant or modified version thereof. In some embodiments, the spaceris a second spacer between the transmembrane domain and a signalingdomain. In some embodiments, the second spacer is an oligopeptide, e.g.,wherein the oligopeptide comprises glycine and serine residues such asbut not limited to glycine-serine doublets. In some embodiments, the CARcomprises two or more spacers, e.g., a spacer between the antigenbinding domain and the transmembrane domain and a spacer between thetransmembrane domain and a signaling domain.

In some embodiments, any one of the cells described herein comprises anucleic acid encoding a CAR or a first generation CAR. In someembodiments, a first generation CAR comprises an antigen binding domain,a transmembrane domain, and signaling domain. In some embodiments, asignaling domain mediates downstream signaling during T cell activation.

In some embodiments, any one of the cells described herein comprises anucleic acid encoding a CAR or a second generation CAR. In someembodiments, a second generation CAR comprises an antigen bindingdomain, a transmembrane domain, and two signaling domains. In someembodiments, a signaling domain mediates downstream signaling during Tcell activation. In some embodiments, a signaling domain is acostimulatory domain. In some embodiments, a costimulatory domainenhances cytokine production, CAR-T cell proliferation, and/or CAR-Tcell persistence during T cell activation.

In some embodiments, any one of the cells described herein comprises anucleic acid encoding a CAR or a third generation CAR. In someembodiments, a third generation CAR comprises an antigen binding domain,a transmembrane domain, and at least three signaling domains. In someembodiments, a signaling domain mediates downstream signaling during Tcell activation. In some embodiments, a signaling domain is acostimulatory domain. In some embodiments, a costimulatory domainenhances cytokine production, CAR-T cell proliferation, and or CAR-Tcell persistence during T cell activation. In some embodiments, a thirdgeneration CAR comprises at least two costimulatory domains. In someembodiments, the at least two costimulatory domains are not the same.

In some embodiments, any one of the cells described herein comprises anucleic acid encoding a CAR or a fourth generation CAR. In someembodiments, a fourth generation CAR comprises an antigen bindingdomain, a transmembrane domain, and at least two, three, or foursignaling domains. In some embodiments, a signaling domain mediatesdownstream signaling during T cell activation. In some embodiments, asignaling domain is a costimulatory domain. In some embodiments, acostimulatory domain enhances cytokine production, CAR-T cellproliferation, and or CAR-T cell persistence during T cell activation.

10. ABD Comprising an Antibody or Antigen-Binding Portion Thereof

In some embodiments, a CAR antigen binding domain is or comprises anantibody or antigen-binding portion thereof. In some embodiments, a CARantigen binding domain is or comprises an scFv or Fab. In someembodiments, a CAR antigen binding domain comprises an scFv or Fabfragment of a CD19 antibody; CD22 antibody; T-cell alpha chain antibody;T-cell β chain antibody; T-cell γ chain antibody; T-cell δ chainantibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7antibody; CD8 antibody; CD11b antibody; CD11c antibody; CD16 antibody;CD20 antibody; CD21 antibody; CD25 antibody; CD28 antibody; CD34antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45ROantibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody;CD80 antibody; CD95 antibody; CD117 antibody; CD127 antibody; CD133antibody; CD137 (4-1 BB) antibody; CD163 antibody; F4/80 antibody;IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARPantibody; LAP antibody; granzyme B antibody; LFA-1 antibody; MR1antibody; uPAR antibody; or transferrin receptor antibody.

In some embodiments, a CAR comprises a signaling domain which is acostimulatory domain. In some embodiments, a CAR comprises a secondcostimulatory domain. In some embodiments, a CAR comprises at least twocostimulatory domains. In some embodiments, a CAR comprises at leastthree costimulatory domains. In some embodiments, a CAR comprises acostimulatory domain selected from one or more of CD27, CD28, 4-1BB,CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83. In some embodiments, if a CAR comprisestwo or more costimulatory domains, two costimulatory domains aredifferent. In some embodiments, if a CAR comprises two or morecostimulatory domains, two costimulatory domains are the same.

In addition to the CARs described herein, various chimeric antigenreceptors and nucleotide sequences encoding the same are known in theart and would be suitable for fusosomal delivery and reprogramming oftarget cells in vivo and in vitro as described herein. See, e.g.,WO2013040557; WO2012079000; WO2016030414; Smith T, et al., NatureNanotechnology. 2017. DOI: 10.1038/NNANO.2017.57, the disclosures ofwhich are herein incorporated by reference.

11. Additional Descriptions of CARs

In certain embodiments, the cell may comprise an exogenouspolynucleotide encoding a CAR. CARs (also known as chimericimmunoreceptors, chimeric T cell receptors, or artificial T cellreceptors) are receptor proteins that have been engineered to give hostcells (e.g., T cells) the new ability to target a specific protein. Thereceptors are chimeric because they combine both antigen-binding and Tcell activating functions into a single receptor. The polycistronicvector of the present disclosure may be used to express one or more CARsin a host cell (e.g., a T cell) for use in cell-based therapies againstvarious target antigens. The CARs expressed by the one or moreexpression cassettes may be the same or different. In these embodiments,the CAR may comprise an extracellular binding domain (also referred toas a “binder”) that specifically binds a target antigen, a transmembranedomain, and an intracellular signaling domain. In certain embodiments,the CAR may further comprise one or more additional elements, includingone or more signal peptides, one or more extracellular hinge domains,and/or one or more intracellular costimulatory domains. Domains may bedirectly adjacent to one another, or there may be one or more aminoacids linking the domains. The nucleotide sequence encoding a CAR may bederived from a mammalian sequence, for example, a mouse sequence, aprimate sequence, a human sequence, or combinations thereof. In thecases where the nucleotide sequence encoding a CAR is non-human, thesequence of the CAR may be humanized. The nucleotide sequence encoding aCAR may also be codon-optimized for expression in a mammalian cell, forexample, a human cell. In any of these embodiments, the nucleotidesequence encoding a CAR may be at least 80% identical (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical) to any of thenucleotide sequences disclosed herein. The sequence variations may bedue to codon-optimalization, humanization, restriction enzyme-basedcloning scars, and/or additional amino acid residues linking thefunctional domains, etc.

In certain embodiments, the CAR may comprise a signal peptide at theN-terminus. Non-limiting examples of signal peptides include CD8a signalpeptide, IgK signal peptide, and granulocyte-macrophagecolony-stimulating factor receptor subunit alpha (GMCSFR-α, also knownas colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signalpeptide, and variants thereof, the amino acid sequences of which areprovided in Table 2 below.

TABLE 2 Exemplary sequences of signal peptides SEQ ID NO: SequenceDescription 6 MALPVTALLLPLALLLHAARP CD8α signal peptide 7METDTLLLWVLLLWVPGSTG IgK signal peptide 8 MLLLVTSLLLCELPHPAFLLIPGMCSFR-α (CSF2RA) signal peptide

In certain embodiments, the extracellular binding domain of the CAR maycomprise one or more antibodies specific to one target antigen ormultiple target antigens. The antibody may be an antibody fragment, forexample, an scFv, or a single-domain antibody fragment, for example, aVHH. In certain embodiments, the scFv may comprise a heavy chainvariable region (VH) and a light chain variable region (V_(L)) of anantibody connected by a linker. The VH and the VL may be connected ineither order, i.e., V_(H)-linker-V_(L) or V_(L)-linker-V_(H).Non-limiting examples of linkers include Whitlow linker, (G₄S)_(n) (ncan be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.) linker, andvariants thereof. In certain embodiments, the antigen may be an antigenthat is exclusively or preferentially expressed on tumor cells, or anantigen that is characteristic of an autoimmune or inflammatory disease.Exemplary target antigens include, but are not limited to, CD5, CD19,CD20, CD22, CD23, CD30, CD70, Kappa, Lambda, and B cell maturation agent(BCMA), G-protein coupled receptor family C group 5 member D (GPRC5D)(associated with leukemias); CS1/SLAMF7, CD38, CD138, GPRC5D, TACI, andBCMA (associated with myelomas); GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA,PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRα, IL-13Rα, Mesothelin,MUC1, MUC16, and ROR1 (associated with solid tumors). In any of theseembodiments, the extracellular binding domain of the CAR can becodon-optimized for expression in a host cell or have variant sequencesto increase functions of the extracellular binding domain.

In certain embodiments, the CAR may comprise a hinge domain, alsoreferred to as a spacer. The terms “hinge” and “spacer” may be usedinterchangeably in the present disclosure. Non-limiting examples ofhinge domains include CD8a hinge domain, CD28 hinge domain, IgG4 hingedomain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acidsequences of which are provided in Table 3 below.

TABLE 3 Exemplary sequences of hinge domains SEQ ID NO: SequenceDescription   9 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGCD8α hinge domain LDFACD  10 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPCD28 hinge domain 113 AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPCD28 hinge domain SKP  11 ESKYGPPCPPCP IgG4 hinge domain  12ESKYGPPCPSCP IgG4 hinge domain  13ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTP IgG4 hinge-EVTCVVVDVSQEDPEVQFNYVDGVEVHNAKTKPREEQFN CH2—CH3 domainSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSSFFLYSRLTVDKSRWQEGNVGSCSVMHEALHNHYTQKSLSLSLGK

In certain embodiments, the transmembrane domain of the CAR may comprisea transmembrane region of the alpha, beta, or zeta chain of a T cellreceptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof,including the human versions of each of these sequences. In otherembodiments, the transmembrane domain may comprise a transmembraneregion of CD8α, CD8β, 4-1BB/CD137, CD28, CD34, CD4, FcεRIγ, CD16,OX40/CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64,CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS,and FGFR2B, or a functional variant thereof, including the humanversions of each of these sequences. Table 4 provides the amino acidsequences of a few exemplary transmembrane domains.

TABLE 4 Exemplary sequences of transmembrane domains SEQ ID NO: SequenceDescription  14 IYIWAPLAGTCGVLLLSLVIT CD8a transmembrane LYC domain  15FWVLVVVGGVLACYSLLVTVA CD28 transmembrane FIIFWV domain 114MFWVLVVVGGVLACYSLLVTV CD28 transmembrane AFIIFWV domain

In certain embodiments, the intracellular signaling domain and/orintracellular costimulatory domain of the CAR may comprise one or moresignaling domains selected from B7-1/CD80, B7-2/CD86, B7-H1/PD-L1,B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4,Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6,4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFFR/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5,DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14,LIGHT/TNFSF14, Lymphotoxin-alpha/TNFβ, OX40/TNFRSF4, OX40 Ligand/TNFSF4,RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNFα, TNF RII/TNFRSF1B,2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2,CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6,SLAM/CD150, CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200,CD300a/LMIR1, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d,Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, TCL1A,TCL1B, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6,TIM-1/KIM-1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associatedantigen-1 (LFA-1), NKG2C, CD3ζ an immunoreceptor tyrosine-basedactivation motif (ITAM), CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40,PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and afunctional variant thereof including the human versions of each of thesesequences. In some embodiments, the intracellular signaling domainand/or intracellular costimulatory domain comprises one or moresignaling domains selected from a CD3ζ domain, an ITAM, a CD28 domain,4-1BB domain, or a functional variant thereof. Table 5 provides theamino acid sequences of a few exemplary intracellular costimulatoryand/or signaling domains. In certain embodiments, as in the case oftisagenlecleucel as described below, the CD3ζ signaling domain of SEQ IDNO:18 may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation,at amino acid position 14 (see SEQ ID NO:115).

TABLE 5Exemplary sequences of intracellular costimulatory and/or signaling domainsSEQ ID NO: Sequence Description 16 KRGRKKLLYIFKQPFMRPVQTTQEEDGC4-1BB costimulatory domain SCRFPEEEEGGCEL 17RSKRSRLLHSDYMNMTPRRPGPTRKHYQ CD28 costimulatory domain PYAPPRDFAAYRS 18RVKFSRSADAPAYQQGQNQLYNELNLGR CD3ζ signaling domainREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR 115 RVKFSRSADAPAYKQGQNQLYNELNLGRCD3ζ signaling domain (with REEYDVLDKRRGRDPEMGGKPRRKNPQQ to K mutation at position EGLYNELQKDKMAEAYSEIGMKGERRR 14)GKGHDGLYQGLSTATKDTYDALHMQAL PPR

In certain embodiments where the polycistronic vector encodes two ormore CARs, the two or more CARs may comprise the same functionaldomains, or one or more different functional domains, as described. Forexample, the two or more CARs may comprise different signal peptides,extracellular binding domains, hinge domains, transmembrane domains,costimulatory domains, and/or intracellular signaling domains, in orderto minimize the risk of recombination due to sequence similarities. Or,alternatively, the two or more CARs may comprise the same domains. Inthe cases where the same domain(s) and/or backbone are used, it isoptional to introduce codon divergence at the nucleotide sequence levelto minimize the risk of recombination.

CD19 CAR

In some embodiments, the CAR is a CD19 CAR (“CD19-CAR”), and in theseembodiments, the polycistronic vector comprises an expression cassettethat contains a nucleotide sequence encoding a CD19 CAR. In someembodiments, the CD19 CAR may comprise a signal peptide, anextracellular binding domain that specifically binds CD19, a hingedomain, a transmembrane domain, an intracellular costimulatory domain,and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the CD19 CAR comprises a CD8asignal peptide. In some embodiments, the CD8a signal peptide comprisesor consists of an amino acid sequence set forth in SEQ ID NO:6 or anamino acid sequence that is at least 80% identical (e.g., at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in of SEQ ID NO:6. In some embodiments, the signal peptidecomprises an IgK signal peptide. In some embodiments, the IgK signalpeptide comprises or consists of an amino acid sequence set forth in SEQID NO:7 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:7. In some embodiments, thesignal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In someembodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consistsof an amino acid sequence set forth in SEQ ID NO:8 or an amino acidsequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical) to the amino acid sequence setforth in of SEQ ID NO:8.

In some embodiments, the extracellular binding domain of the CD19 CAR isspecific to CD19, for example, human CD19. The extracellular bindingdomain of the CD19 CAR can be codon-optimized for expression in a hostcell or to have variant sequences to increase functions of theextracellular binding domain. In some embodiments, the extracellularbinding domain comprises an immunogenically active portion of animmunoglobulin molecule, for example, an scFv.

In some embodiments, the extracellular binding domain of the CD19 CARcomprises an scFv derived from the FMC63 monoclonal antibody (FMC63),which comprises the heavy chain variable region (V_(H)) and the lightchain variable region (V_(L)) of FMC63 connected by a linker. FMC63 andthe derived scFv have been described in Nicholson et al., Mol. Immun.34(16-17):1157-1165 (1997) and PCT Application Publication No.WO2018/213337, the entire contents of each of which are incorporated byreference herein. In some embodiments, the amino acid sequences of theentire FMC63-derived scFv (also referred to as FMC63 scFv) and itsdifferent portions are provided in Table 6 below. In some embodiments,the CD19-specific scFv comprises or consists of an amino acid sequenceset forth in SEQ ID NO:19, 20, or 25, or an amino acid sequence that isat least 80% identical (e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in SEQ ID NO:19,20, or 25. In some embodiments, the CD19-specific scFv may comprise oneor more CDRs having amino acid sequences set forth in SEQ ID NOs: 21-23and 26-28. In some embodiments, the CD19-specific scFv may comprise alight chain with one or more CDRs having amino acid sequences set forthin SEQ ID NOs: 21-23. In some embodiments, the CD19-specific scFv maycomprise a heavy chain with one or more CDRs having amino acid sequencesset forth in SEQ ID NOs: 26-28. In any of these embodiments, theCD19-specific scFv may comprise one or more CDRs comprising one or moreamino acid substitutions, or comprising a sequence that is at least 80%identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical), to any of the sequences identified. In some embodiments, theextracellular binding domain of the CD19 CAR comprises or consists ofthe one or more CDRs as described herein.

In some embodiments, the linker linking the VH and the VL portions ofthe scFv is a Whitlow linker having an amino acid sequence set forth inSEQ ID NO:24. In some embodiments, the Whitlow linker may be replaced bya different linker, for example, a 3xG₄S linker having an amino acidsequence set forth in SEQ ID NO:30, which gives rise to a differentFMC63-derived scFv having an amino acid sequence set forth in SEQ IDNO:29. In certain of these embodiments, the CD19-specific scFv comprisesor consists of an amino acid sequence set forth in SEQ ID NO:29 or anamino acid sequence that is at least 80% identical (e.g., at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in of SEQ ID NO:29.

TABLE 6 Exemplary sequences of anti-CD19 scFv and components SEQ ID NO:Amino Acid Sequence Description 19 DIQMTQTTSSLSASLGDRVTISCRASAnti-CD19 FMC63 scFv QDISKYLNWYQQKPDGTVKLLIYHT entire sequence,SRLHSGVPSRFSGSGSGTDYSLTISN with Whitlow linker LEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVK LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS YAMDYWGQGTSVTVSS 20 DIQMTQTTSSLSASLGDRVTISCRASAnti-CD19 FMC63 scFv QDISKYLNWYQQKPDGTVKLLIYHT light chain variableSRLHSGVPSRFSGSGSGTDYSLTISN region LEQEDIATYFCQQGNTLPYTFGGGT KLEIT 21QDISKY Anti-CD19 FMC63 scFv light chain CDR1 22 HTS Anti-CD19 FMC63 scFvlight chain CDR2 23 QQGNTLPYT Anti-CD19 FMC63 scFv light chain CDR3 24GSTSGSGKPGSGEGSTKG Whitlow linker 25 EVKLQESGPGLVAPSQSLSVTCTVSAnti-CD19 FMC63 scFv GVSLPDYGVSWIRQPPRKGLEWLG heavy chain variableVIWGSETTYYNSALKSRLTIIKDNSK region SQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 26 GVSLPDYG Anti-CD19 FMC63 scFv heavy chain CDR127 IWGSETT Anti-CD19 FMC63 scFv heavy chain CDR2 28 AKHYYYGGSYAMDYAnti-CD19 FMC63 scFv heavy chain CDR3 29 DIQMTQTTSSLSASLGDRVTISCRASAnti-CD19 FMC63 scFv QDISKYLNWYQQKPDGTVKLLIYHT entire sequence, SRLHSGVPSRFSGSGSGTDYSLTISN with 3xG₄S linker LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY AMDYWGQGTSVTVSS 30 GGGGSGGGGSGGGGS 3xG₄S linker

In some embodiments, the extracellular binding domain of the CD19 CAR isderived from an antibody specific to CD19, including, for example,SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), H1D37 (Pezuttoet al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al.,Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)),B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J.Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther.335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10):2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol.118:368-381(1989)). In any of these embodiments, the extracellularbinding domain of the CD19 CAR can comprise or consist of the V_(H), theV_(L), and/or one or more CDRs of any of the antibodies.

In some embodiments, the hinge domain of the CD19 CAR comprises a CD8ahinge domain, for example, a human CD8a hinge domain. In someembodiments, the CD8a hinge domain comprises or consists of an aminoacid sequence set forth in SEQ ID NO:9 or an amino acid sequence that isat least 80% identical (e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:9.In some embodiments, the hinge domain comprises a CD28 hinge domain, forexample, a human CD28 hinge domain. In some embodiments, the CD28 hingedomain comprises or consists of an amino acid sequence set forth in SEQID NO:10 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:10. In some embodiments, thehinge domain comprises an IgG4 hinge domain, for example, a human IgG4hinge domain. In some embodiments, the IgG4 hinge domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:11 or SEQ IDNO:12, or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:11 or SEQ ID NO:12. In someembodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, forexample, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, theIgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acidsequence set forth in SEQ ID NO:13 or an amino acid sequence that is atleast 80% identical (e.g., at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:13.

In some embodiments, the transmembrane domain of the CD19 CAR comprisesa CD8a transmembrane domain, for example, a human CD8a transmembranedomain. In some embodiments, the CD8a transmembrane domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:14 or an aminoacid sequence that is at least 80% identical (e.g., at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in SEQ ID NO:14. In some embodiments, the transmembrane domaincomprises a CD28 transmembrane domain, for example, a human CD28transmembrane domain. In some embodiments, the CD28 transmembrane domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:15 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:15.

In some embodiments, the intracellular costimulatory domain of the CD19CAR comprises a 4-1BB costimulatory domain. 4-1BB, also known as CD137,transmits a potent costimulatory signal to T cells, promotingdifferentiation and enhancing long-term survival of T lymphocytes. Insome embodiments, the 4-1BB costimulatory domain is human. In someembodiments, the 4-1BB costimulatory domain comprises or consists of anamino acid sequence set forth in SEQ ID NO:16 or an amino acid sequencethat is at least 80% identical (e.g., at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical) to the amino acid sequence set forth inSEQ ID NO:16. In some embodiments, the intracellular costimulatorydomain comprises a CD28 costimulatory domain. CD28 is anotherco-stimulatory molecule on T cells. In some embodiments, the CD28costimulatory domain is human. In some embodiments, the CD28costimulatory domain comprises or consists of an amino acid sequence setforth in SEQ ID NO:17 or an amino acid sequence that is at least 80%identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical) to the amino acid sequence set forth in SEQ ID NO:17. In someembodiments, the intracellular costimulatory domain of the CD19 CARcomprises a 4-1BB costimulatory domain and a CD28 costimulatory domainas described.

In some embodiments, the intracellular signaling domain of the CD19 CARcomprises a CD3 zeta (ζ) signaling domain. CD3ζ associates with T cellreceptors (TCRs) to produce a signal and contains immunoreceptortyrosine-based activation motifs (ITAMs). The CD3ζ signaling domainrefers to amino acid residues from the cytoplasmic domain of the zetachain that are sufficient to functionally transmit an initial signalnecessary for T cell activation. In some embodiments, the CD3ζ signalingdomain is human. In some embodiments, the CD3ζ signaling domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:18 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:18.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD19 CAR,including, for example, a CD19 CAR comprising the CD19-specific scFvhaving sequences set forth in SEQ ID NO:19 or SEQ ID NO:29, the CD8αhinge domain of SEQ ID NO:9, the CD8α transmembrane domain of SEQ IDNO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3ζsignaling domain of SEQ ID NO:18, and/or variants (i.e., having asequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.In any of these embodiments, the CD19 CAR may additionally comprise asignal peptide (e.g., a CD8α signal peptide) as described.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD19 CAR,including, for example, a CD19 CAR comprising the CD19-specific scFvhaving sequences set forth in SEQ ID NO:19 or SEQ ID NO:29, the IgG4hinge domain of SEQ ID NO:11 or SEQ ID NO:12, the CD28 transmembranedomain of SEQ ID NO:15, the 4-1BB costimulatory domain of SEQ ID NO:16,the CD3ζ signaling domain of SEQ ID NO:18, and/or variants (i.e., havinga sequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.In any of these embodiments, the CD19 CAR may additionally comprise asignal peptide (e.g., a CD8α signal peptide) as described.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD19 CAR,including, for example, a CD19 CAR comprising the CD19-specific scFvhaving sequences set forth in SEQ ID NO:19 or SEQ ID NO:29, the CD28hinge domain of SEQ ID NO:10, the CD28 transmembrane domain of SEQ IDNO:15, the CD28 costimulatory domain of SEQ ID NO:17, the CD3ζ signalingdomain of SEQ ID NO:18, and/or variants (i.e., having a sequence that isat least 80% identical, for example, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99 identical to the disclosed sequence) thereof. In any of theseembodiments, the CD19 CAR may additionally comprise a signal peptide(e.g., a CD8α signal peptide) as described.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD19 CAR as setforth in SEQ ID NO:116 or is at least 80% identical (e.g., at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the nucleotide sequenceset forth in SEQ ID NO:116 (see Table 7). The encoded CD19 CAR has acorresponding amino acid sequence set forth in SEQ ID NO:117 or is atleast 80% identical (e.g., at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ IDNO:117, with the following components: CD8α signal peptide, FMC63 scFv(V_(L)-Whitlow linker-V_(H)), CD8α hinge domain, CD8α transmembranedomain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a commerciallyavailable embodiment of CD19 CAR. Non-limiting examples of commerciallyavailable embodiments of CD19 CARs expressed and/or encoded by T cellsinclude tisagenlecleucel, lisocabtagene maraleucel, axicabtageneciloleucel, and brexucabtagene autoleucel.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding tisagenlecleucelor portions thereof. Tisagenlecleucel comprises a CD19 CAR with thefollowing components: CD8α signal peptide, FMC63 scFv (VL-3xG₄Slinker-V_(H)), CD8α hinge domain, CD8α transmembrane domain, 4-1BBcostimulatory domain, and CD3ζ signaling domain. The nucleotide andamino acid sequence of the CD19 CAR in tisagenlecleucel are provided inTable 7, with annotations of the sequences provided in Table 8.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding lisocabtagenemaraleucel or portions thereof. Lisocabtagene maraleucel comprises aCD19 CAR with the following components: GMCSFR-α or CSF2RA signalpeptide, FMC63 scFv (V_(L)-Whitlow linker-V_(H)), IgG4 hinge domain,CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3ζsignaling domain. The nucleotide and amino acid sequence of the CD19 CARin lisocabtagene maraleucel are provided in Table 7, with annotations ofthe sequences provided in Table 9.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding axicabtageneciloleucel or portions thereof. Axicabtagene ciloleucel comprises a CD19CAR with the following components: GMCSFR-α or CSF2RA signal peptide,FMC63 scFv (VL-Whitlow linker-VH), CD28 hinge domain, CD28 transmembranedomain, CD28 costimulatory domain, and CD3ζ signaling domain. Thenucleotide and amino acid sequence of the CD19 CAR in axicabtageneciloleucel are provided in Table 7, with annotations of the sequencesprovided in Table 10.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding brexucabtageneautoleucel or portions thereof. Brexucabtagene autoleucel comprises aCD19 CAR with the following components: GMCSFR-α signal peptide, FMC63scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatorydomain, and CD3ζ signaling domain.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD19 CAR as setforth in SEQ ID NO: 31, 33, or 35, or is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to thenucleotide sequence set forth in SEQ ID NO: 31, 33, or 35. The encodedCD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO:32, 34, or 36, respectively, or is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO: 32, 34, or 36, respectively.

TABLE 7 Exemplary sequences of CD19 CARs SEQ ID NO: Sequence Description116 atggccttaccagtgaccgccttgctcctgccgctggccttgctgct Exemplary CD19ccacgccgccaggccggacatccagatgacacagactacatcctc CAR nucleotidecctgtctgcctctctgggagacagagtcaccatcagttgcagggca sequenceagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccc cctcgc 117MALPVTALLLPLALLLHAARPDIQMTQTTS Exemplary CD19SLSASLGDRVTISCRASQDISKYLNWYQQK CAR amino acidPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT sequence DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKL QESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 31 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctTisagenlecleucel ccacgccgccaggccggacatccagatgacacagactacatcctc CD19 CARcctgtctgcctctctgggagacagagtcaccatcagttgcagggca nucleotideagtcaggacattagtaaatatttaaattggtatcagcagaaaccagat sequenceggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcg c 32MALPVTALLLPLALLLHAARPDIQMTQTTS TisagenlecleucelSLSASLGDRVTISCRASQDISKYLNWYQQK CD19 CAR aminoPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT acid sequenceDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCA KHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR 33atgctgctgctggtgaccagcctgctgctgtgcgagctgccccacc Lisocabtageneccgcctttctgctgatccccgacatccagatgacccagaccacctc maraleucel CD19cagcctgagcgccagcctgggcgaccgggtgaccatcagctgcc CAR nucleotidegggccagccaggacatcagcaagtacctgaactggtatcagcag sequenceaagcccgacggcaccgtcaagctgctgatctaccacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagcgaatctaagtacggaccgccctgccccccttgccctatgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtgaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgcgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcagaagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccaggaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccccaag g 34MLLLVTSLLLCELPHPAFLLIPDIQMTQTTS LisocabtageneSLSASLGDRVTISCRASQDISKYLNWYQQK maraleucel CD19PDGTVKLLIYHTSRLHSGVPSRFSGSGSGT CAR amino acidDYSLTISNLEQEDIATYFCQQGNTLPYTFG sequence GGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLL VTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 35 atgcttctcctggtgacaagccttctgctctgtgagttaccacacccaAxicabtagene gcattcctcctgatcccagacatccagatgacacagactacatcctcciloleucel CD19 cctgtctgcctctctgggagacagagtcaccatcagttgcagggcaCAR nucleotide agtcaggacattagtaaatatttaaattggtatcagcagaaaccagat sequenceggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc 36MLLLVTSLLLCELPHPAFLLIPDIQMTQTTS AxicabtageneSLSASLGDRVTISCRASQDISKYLNWYQQK ciloleucel CD19PDGTVKLLIYHTSRLHSGVPSRFSGSGSGT CAR amino acidDYSLTISNLEQEDIATYFCQQGNTLPYTFG sequence GGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLC PSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR

TABLE 8 Annotation of tisagenlecleucel CD19 CAR sequences NucleotideAmino Sequence Acid Sequence Feature Position Position CD8α signalpeptide  1-63  1-21 FMC63 scFv  64-789  22-263 (V_(L)-3xG₄Slinker-V_(H)) CD8α hinge domain 790-924 264-308 CD8α transmembranedomain 925-996 309-332 4-1BB costimulatory domain  997-1122 333-374 CD3ζsignaling domain 1123-1458 375-486

TABLE 9 Annotation of lisocabtagene maraleucel CD19 CAR sequencesNucleotide Amino Sequence Acid Sequence Feature Position PositionGMCSFR-α signal peptide  1-66  1-22 FMC63 scFv  67-801  23-267(V_(L)-Whitlow linker-V_(H)) IgG4 hinge domain 802-837 268-279 CD28transmembrane domain 838-921 280-307 4-1BB costimulatory domain 922-1047 308-349 CD3ζ signaling domain 1048-1383 350-461

TABLE 10 Annotation of axicabtagene ciloleucel CD19 CAR sequencesNucleotide Amino Sequence Acid Sequence Feature Position Position CSF2RAsignal peptide  1-66  1-22 FMC63 scFv  67-801  23-267 (V_(L)-Whitlowlinker-V_(H)) CD28 hinge domain 802-927 268-309 CD28 transmembranedomain  928-1008 310-336 CD28 costimulatory domain 1009-1131 337-377CD3ζ signaling domain 1132-1467 378-489

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding CD19 CAR as setforth in SEQ ID NO: 31, 33, or 35, or at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to thenucleotide sequence set forth in SEQ ID NO: 31, 33, or 35. The encodedCD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO:32, 34, or 36, respectively, is at least 80% identical (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical) to the amino acidsequence set forth in of SEQ ID NO: 32, 34, or 36, respectively.

CD20 CAR

In some embodiments, the CAR is a CD20 CAR (“CD20-CAR”), and in theseembodiments, the polycistronic vector comprises an expression cassettethat contains a nucleotide sequence encoding a CD20 CAR. CD20 is anantigen found on the surface of B cells as early at the pro-B phase andprogressively at increasing levels until B cell maturity, as well as onthe cells of most B-cell neoplasms. CD20 positive cells are alsosometimes found in cases of Hodgkins disease, myeloma, and thymoma. Insome embodiments, the CD20 CAR may comprise a signal peptide, anextracellular binding domain that specifically binds CD20, a hingedomain, a transmembrane domain, an intracellular costimulatory domain,and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the CD20 CAR comprises a CD8αsignal peptide. In some embodiments, the CD8α signal peptide comprisesor consists of an amino acid sequence set forth in SEQ ID NO:6 or anamino acid sequence that is at least 80% identical (e.g., at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in of SEQ ID NO:6. In some embodiments, the signal peptidecomprises an IgK signal peptide. In some embodiments, the IgK signalpeptide comprises or consists of an amino acid sequence set forth in SEQID NO:7 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:7. In some embodiments, thesignal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In someembodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consistsof an amino acid sequence set forth in SEQ ID NO:8 or an amino acidsequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical) to the amino acid sequence setforth in of SEQ ID NO:8.

In some embodiments, the extracellular binding domain of the CD20 CAR isspecific to CD20, for example, human CD20. The extracellular bindingdomain of the CD20 CAR can be codon-optimized for expression in a hostcell or to have variant sequences to increase functions of theextracellular binding domain. In some embodiments, the extracellularbinding domain comprises an immunogenically active portion of animmunoglobulin molecule, for example, an scFv.

In some embodiments, the extracellular binding domain of the CD20 CAR isderived from an antibody specific to CD20, including, for example,Leu16, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab,tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. Inany of these embodiments, the extracellular binding domain of the CD20CAR can comprise or consist of the VH, the VL, and/or one or more CDRsof any of the antibodies.

In some embodiments, the extracellular binding domain of the CD20 CARcomprises an scFv derived from the Leu16 monoclonal antibody, whichcomprises the heavy chain variable region (VH) and the light chainvariable region (VL) of Leu16 connected by a linker. See Wu et al.,Protein Engineering. 14(12):1025-1033 (2001). In some embodiments, thelinker is a 3xG₄S linker. In other embodiments, the linker is a Whitlowlinker as described herein. In some embodiments, the amino acidsequences of different portions of the entire Leu16-derived scFv (alsoreferred to as Leu16 scFv) and its different portions are provided inTable 11 below. In some embodiments, the CD20-specific scFv comprises orconsists of an amino acid sequence set forth in SEQ ID NO:37, 38, or 42,or an amino acid sequence that is at least 80% identical (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical) to the amino acidsequence set forth in of SEQ ID NO:37, 38, or 42. In some embodiments,the CD20-specific scFv may comprise one or more CDRs having amino acidsequences set forth in SEQ ID NOs: 39-41, 43 and 44. In someembodiments, the CD20-specific scFv may comprise a light chain with oneor more CDRs having amino acid sequences set forth in SEQ ID NOs: 39-41.In some embodiments, the CD20-specific scFv may comprise a heavy chainwith one or more CDRs having amino acid sequences set forth in SEQ IDNOs: 43-44. In any of these embodiments, the CD20-specific scFv maycomprise one or more CDRs comprising one or more amino acidsubstitutions, or comprising a sequence that is at least 80% identical(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical), toany of the sequences identified. In some embodiments, the extracellularbinding domain of the CD20 CAR comprises or consists of the one or moreCDRs as described herein.

TABLE 11 Exemplary sequences of anti-CD20 scFv and components SEQ ID NO:Amino Acid Sequence Description 37 DIVLTQSPAILSASPGEKVTMTCRAAnti-CD20 Leu 16 scFv SSSVNYMDWYQKKPGSSPKPWIYAT entire sequence, withSNLASGVPARFSGSGSGTSYSLTIS Whitlow linker 4VEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGSGKPGSGEGSTKGE VQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGA IYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSN YYGSSYWFFDVWGAGTTVTVSS 38DIVLTQSPAILSASPGEKVTMTCRA Anti-CD20 Leu16 scFv SSSVNYMDWYQKKPGSSPKPWIYATlight chain variable SNLASGVPARFSGSGSGTSYSLTIS regionRVEAEDAATYYCQQWSFNPPTFGGG TKLEIK 39 RASSSVNYMD Anti-CD20 Leu16 scFvlight chain CDR1 40 ATSNLAS Anti-CD20 Leu16 scFv light chain CDR2 41QQWSFNPPT Anti-CD20 Leu16 scFv light chain CDR3 42EVQLQQSGAELVKPGASVKMSCKA Anti-CD20 Leu16 scFv SGYTFTSYNMHWVKQTPGQGLEWIheavy chain GAIYPGNGDTSYNQKFKGKATLTA DKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVS S 43 SYNMH Anti-CD20 Leu16 scFvheavy chain CDR1 44 AIYPGNGDTSYNQKFKG Anti-CD20 Leu16 scFvheavy chain CDR2

In some embodiments, the hinge domain of the CD20 CAR comprises a CD8αhinge domain, for example, a human CD8α hinge domain. In someembodiments, the CD8α hinge domain comprises or consists of an aminoacid sequence set forth in SEQ ID NO:9 or an amino acid sequence that isat least 80% identical (e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:9.In some embodiments, the hinge domain comprises a CD28 hinge domain, forexample, a human CD28 hinge domain. In some embodiments, the CD28 hingedomain comprises or consists of an amino acid sequence set forth in SEQID NO: 10 or an amino acid sequence that is at least 80% identical(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical) to theamino acid sequence set forth in of SEQ TD NO: 10. In some embodiments,the hinge domain comprises an IgG4 hinge domain, for example, a humanIgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprisesor consists of an amino acid sequence set forth in SEQ ID NO:11 or SEQID NO:12, or an amino acid sequence that is at least 80% identical(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical) to theamino acid sequence set forth in of SEQ ID NO:11 or SEQ ID NO:12. Insome embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In someembodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of anamino acid sequence set forth in SEQ ID NO:13 or an amino acid sequencethat is at least 80% identical (e.g., at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical) to the amino acid sequence set forth in ofSEQ ID NO:13.

In some embodiments, the transmembrane domain of the CD20 CAR comprisesa CD8α transmembrane domain, for example, a human CD8α transmembranedomain. In some embodiments, the CD8α transmembrane domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:14 or an aminoacid sequence that is at least 80% identical (e.g., at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in SEQ ID NO:14. In some embodiments, the transmembrane domaincomprises a CD28 transmembrane domain, for example, a human CD28transmembrane domain. In some embodiments, the CD28 transmembrane domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:15 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:15.

In some embodiments, the intracellular costimulatory domain of the CD20CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BBcostimulatory domain. In some embodiments, the 4-1BB costimulatorydomain comprises or consists of an amino acid sequence set forth in SEQID NO:16 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:16. In some embodiments, theintracellular costimulatory domain comprises a CD28 costimulatorydomain, for example, a human CD28 costimulatory domain. In someembodiments, the CD28 costimulatory domain comprises or consists of anamino acid sequence set forth in SEQ ID NO:17 or an amino acid sequencethat is at least 80% identical (e.g., at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical) to the amino acid sequence set forth inSEQ ID NO:17.

In some embodiments, the intracellular signaling domain of the CD20 CARcomprises a CD3 zeta (ζ) signaling domain, for example, a human CD3ζsignaling domain. In some embodiments, the CD3ζ signaling domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:18 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:18.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD20 CAR,including, for example, a CD20 CAR comprising the CD20-specific scFvhaving sequences set forth in SEQ ID NO:37, the CD8α hinge domain of SEQID NO:9, the CD8α transmembrane domain of SEQ ID NO:14, the 4-1BBcostimulatory domain of SEQ ID NO:16, the CD3ζ signaling domain of SEQID NO:18, and/or variants (i.e., having a sequence that is at least 80%identical, for example, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD20 CAR,including, for example, a CD20 CAR comprising the CD20-specific scFvhaving sequences set forth in SEQ ID NO:37, the CD28 hinge domain of SEQID NO:10, the CD8α transmembrane domain of SEQ ID NO:14, the 4-BBcostimulatory domain of SEQ ID NO:16, the CD3ζ signaling domain of SEQID NO:18, and/or variants (i.e., having a sequence that is at least 80%identical, for example, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD20 CAR,including, for example, a CD20 CAR comprising the CD20-specific scFvhaving sequences set forth in SEQ ID NO:37, the IgG4 hinge domain of SEQID NO:11 or SEQ ID NO:12, the CD8α transmembrane domain of SEQ ID NO:14,the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3ζ signalingdomain of SEQ ID NO:18, and/or variants (i.e., having a sequence that isat least 80% identical, for example, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99 identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD20 CAR,including, for example, a CD20 CAR comprising the CD20-specific scFvhaving sequences set forth in SEQ ID NO:37, the CD8α hinge domain of SEQID NO:9, the CD28 transmembrane domain of SEQ ID NO:15, the 4-1BBcostimulatory domain of SEQ ID NO:16, the CD3ζ signaling domain of SEQID NO:18, and/or variants (i.e., having a sequence that is at least 80%identical, for example, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD20 CAR,including, for example, a CD20 CAR comprising the CD20-specific scFvhaving sequences set forth in SEQ ID NO:37, the CD28 hinge domain of SEQID NO:10, the CD28 transmembrane domain of SEQ ID NO:15, the 4-1BBcostimulatory domain of SEQ ID NO:16, the CD3ζ signaling domain of SEQID NO:18, and/or variants (i.e., having a sequence that is at least 80%identical, for example, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD20 CAR,including, for example, a CD20 CAR comprising the CD20-specific scFvhaving sequences set forth in SEQ ID NO:37, the IgG4 hinge domain of SEQID NO:11 or SEQ ID NO:1, the CD28 transmembrane domain of SEQ ID NO:15,the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3ζ signalingdomain of SEQ ID NO:18, and/or variants (i.e., having a sequence that isat least 80% identical, for example, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99 identical to the disclosed sequence) thereof.

CD22 CAR

In some embodiments, the CAR is a CD22 CAR (“CD22-CAR”), and in theseembodiments, the polycistronic vector comprises an expression cassettethat contains a nucleotide sequence encoding a CD22 CAR. CD22, which isa transmembrane protein found mostly on the surface of mature B cellsthat functions as an inhibitory receptor for B cell receptor (BCR)signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias(e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acutelymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not presenton the cell surface in early stages of B cell development or on stemcells. In some embodiments, the CD22 CAR may comprise a signal peptide,an extracellular binding domain that specifically binds CD22, a hingedomain, a transmembrane domain, an intracellular costimulatory domain,and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the CD22 CAR comprises a CD8αsignal peptide. In some embodiments, the CD8α signal peptide comprisesor consists of an amino acid sequence set forth in SEQ ID NO:6 or anamino acid sequence that is at least 80% identical (e.g., at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in of SEQ ID NO:6. In some embodiments, the signal peptidecomprises an IgK signal peptide. In some embodiments, the IgK signalpeptide comprises or consists of an amino acid sequence set forth in SEQID NO:7 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:7. In some embodiments, thesignal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In someembodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consistsof an amino acid sequence set forth in SEQ ID NO:8 or an amino acidsequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical) to the amino acid sequence setforth in of SEQ ID NO:8.

In some embodiments, the extracellular binding domain of the CD22 CAR isspecific to CD22, for example, human CD22. The extracellular bindingdomain of the CD22 CAR can be codon-optimized for expression in a hostcell or to have variant sequences to increase functions of theextracellular binding domain. In some embodiments, the extracellularbinding domain comprises an immunogenically active portion of animmunoglobulin molecule, for example, an scFv.

In some embodiments, the extracellular binding domain of the CD22 CAR isderived from an antibody specific to CD22, including, for example, SM03,inotuzumab, epratuzumab, moxetumomab, and pinatuzumab. In any of theseembodiments, the extracellular binding domain of the CD22 CAR cancomprise or consist of the V_(H), the V_(L), and/or one or more CDRs ofany of the antibodies.

In some embodiments, the extracellular binding domain of the CD22 CARcomprises an scFv derived from the m971 monoclonal antibody (m971),which comprises the heavy chain variable region (V_(H)) and the lightchain variable region (V_(L)) of m971 connected by a linker. In someembodiments, the linker is a 3xG₄S linker. In other embodiments, theWhitlow linker may be used instead. In some embodiments, the amino acidsequences of the entire m971-derived scFv (also referred to as m971scFv) and its different portions are provided in Table 12 below. In someembodiments, the CD22-specific scFv comprises or consists of an aminoacid sequence set forth in SEQ ID NO:45, 46, or 50, or an amino acidsequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical) to the amino acid sequence setforth in of SEQ ID NO:45, 46, or 50. In some embodiments, theCD22-specific scFv may comprise one or more CDRs having amino acidsequences set forth in SEQ ID NOs: 47-49 and 51-53. In some embodiments,the CD22-specific scFv may comprise a heavy chain with one or more CDRshaving amino acid sequences set forth in SEQ ID NOs: 47-49. In someembodiments, the CD22-specific scFv may comprise a light chain with oneor more CDRs having amino acid sequences set forth in SEQ ID NOs: 51-53.In any of these embodiments, the CD22-specific scFv may comprise one ormore CDRs comprising one or more amino acid substitutions, or comprisinga sequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical), to any of the sequencesidentified. In some embodiments, the extracellular binding domain of theCD22 CAR comprises or consists of the one or more CDRs as describedherein.

In some embodiments, the extracellular binding domain of the CD22 CARcomprises an scFv derived from m971-L7, which is an affinity maturedvariant of m971 with significantly improved CD22 binding affinitycompared to the parental antibody m971 (improved from about 2 nM to lessthan 50 pM). In some embodiments, the scFv derived from m971-L7comprises the V_(H) and the V_(L) of m971-L7 connected by a 3xG₄Slinker. In other embodiments, the Whitlow linker may be used instead. Insome embodiments, the amino acid sequences of the entire m971-L7-derivedscFv (also referred to as m971-L7 scFv) and its different portions areprovided in Table 12 below. In some embodiments, the CD22-specific scFvcomprises or consists of an amino acid sequence set forth in SEQ IDNO:54, 55, or 59, or an amino acid sequence that is at least 80%identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical) to the amino acid sequence set forth in of SEQ ID NO:54, 55,or 59. In some embodiments, the CD22-specific scFv may comprise one ormore CDRs having amino acid sequences set forth in SEQ ID NOs: 56-58 and60-62. In some embodiments, the CD22-specific scFv may comprise a heavychain with one or more CDRs having amino acid sequences set forth in SEQID NOs: 56-58. In some embodiments, the CD22-specific scFv may comprisea light chain with one or more CDRs having amino acid sequences setforth in SEQ ID NOs: 60-62. In any of these embodiments, theCD22-specific scFv may comprise one or more CDRs comprising one or moreamino acid substitutions, or comprising a sequence that is at least 80%identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical), to any of the sequences identified. In some embodiments, theextracellular binding domain of the CD22 CAR comprises or consists ofthe one or more CDRs as described herein.

TABLE 12 Exemplary sequences of anti-CD22 scFv and components SEQ ID NO:Amino Acid Sequence Description 45 QVQLQQSGPGLVKPSQTLSLTCAISGAnti-CD22 m971 scFv DSVSSNSAAWNWIRQSPSRGLEWL entire sequence, withGRTYYRSKWYNDYAVSVKSRITINP 3xG₄S linker DTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNW YQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATY YCQQSYSIPQTFGQGTKLEIK 46QVQLQQSGPGLVKPSQTLSLTCAISG Anti-CD22 m971 scFv DSVSSNSAAWNWIRQSPSRGLEWLheavy chain variable GRTYYRSKWYNDYAVSVKSRITINP regionDTSKNQFSLQLNSVTPEDTAVYYCA REVTGDLEDAFDIWGQGTMVTVSS 47 GDSVSSNSAAAnti-CD22 m971 scFv heavy chain CDR1 48 TYYRSKWYN Anti-CD22 m971 scFvheavy chain CDR2 49 AREVTGDLEDAFDI Anti-CD22 m971 scFv heavy chain CDR350 DIQMTQSPSSLSASVGDRVTITCRAS Anti-CD22 m971 scFvQTIWSYLNWYQQRPGKAPNLLIYA light chain ASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGT KLEIK 51 QTIWSY Anti-CD22 m971 scFvlight chain CDR1 52 AAS Anti-CD22 m971 scFv light chain CDR2 53QQSYSIPQT Anti-CD22 m971 scFv light chain CDR3 54QVQLQQSGPGMVKPSQTLSLTCAIS Anti-CD22 m971-L7 GDSVSSNSVAWNWIRQSPSRGLEWscFv entire sequence, LGRTYYRSTWYNDYAVSMKSRITIN with 3xG4S linkerPDTNKNQFSLQLNSVTPEDTAVYYC AREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMIQSPS SLSASVGDRVTITCRASQTIWSYLNWYRQRPGEAPNLLIYAASSLQSGVP SRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK 55 QVQLQQSGPGMVKPSQTLSLTCAIS Anti-CD22 m971-L7GDSVSSNSVAWNWIRQSPSRGLEW scFv heavy chain LGRTYYRSTWYNDYAVSMKSRITINvariable region PDTNKNQFSLQLNSVTPEDTAVYYC AREVTGDLEDAFDIWGQGTMVTVS S 56GDSVSSNSVA Anti-CD22 m971-L7 scFv heavy chain CDR1 57 TYYRSTWYNAnti-CD22 m971-L7 scFv heavy chain CDR2 58 AREVTGDLEDAFDIAnti-CD22 m971-L7 scFv heavy chain CDR3 59 DIQMIQSPSSLSASVGDRVTITCRASAnti-CD22 m971-L7 QTIWSYLNWYRQRPGEAPNLLIYAA scFv light chain variableSSLQSGVPSRFSGRGSGTDFTLTISSL region QAEDFATYYCQQSYSIPQTFGQGTK LEIK 60QTIWSY Anti-CD22 m971-L7 scFv light chain CDR1 61 AAS Anti-CD22 m971-L7scFv light chain CDR2 62 QQSYSIPQT Anti-CD22 m971-L7scFv light chain CDR3

In some embodiments, the extracellular binding domain of the CD22 CARcomprises immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 aretherapeutic agents that comprise an scFv specific for CD22 fused to abacterial toxin, and thus can bind to the surface of the cancer cellsthat express CD22 and kill the cancer cells. BL22 comprises a dsFv of ananti-CD22 antibody, RFB4, fused to a 38-kDa truncated form ofPseudomonas exotoxin A (Bang et al., Clin. Cancer Res., 11:1545-50(2005)). HA22 (CAT8015, moxetumomab pasudotox) is a mutated, higheraffinity version of BL22 (Ho et al., J. Biol. Chem., 280(1): 607-17(2005)). Suitable sequences of antigen binding domains of HA22 and BL22specific to CD22 are disclosed in, for example, U.S. Pat. Nos.7,541,034; 7,355,012; and 7,982,011, which are hereby incorporated byreference in their entirety.

In some embodiments, the hinge domain of the CD22 CAR comprises a CD8αhinge domain, for example, a human CD8α hinge domain. In someembodiments, the CD8α hinge domain comprises or consists of an aminoacid sequence set forth in SEQ ID NO:9 or an amino acid sequence that isat least 80% identical (e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:9.In some embodiments, the hinge domain comprises a CD28 hinge domain, forexample, a human CD28 hinge domain. In some embodiments, the CD28 hingedomain comprises or consists of an amino acid sequence set forth in SEQID NO:10 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:10. In some embodiments, thehinge domain comprises an IgG4 hinge domain, for example, a human IgG4hinge domain. In some embodiments, the IgG4 hinge domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:11 or SEQ IDNO:12, or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:11 or SEQ ID NO:12. In someembodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, forexample, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, theIgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acidsequence set forth in SEQ ID NO:13 or an amino acid sequence that is atleast 80% identical (e.g., at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:13.

In some embodiments, the transmembrane domain of the CD22 CAR comprisesa CD8α transmembrane domain, for example, a human CD8α transmembranedomain. In some embodiments, the CD8α transmembrane domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:14 or an aminoacid sequence that is at least 80% identical (e.g., at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in SEQ ID NO:14. In some embodiments, the transmembrane domaincomprises a CD28 transmembrane domain, for example, a human CD28transmembrane domain. In some embodiments, the CD28 transmembrane domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:15 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:15.

In some embodiments, the intracellular costimulatory domain of the CD22CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BBcostimulatory domain. In some embodiments, the 4-1BB costimulatorydomain comprises or consists of an amino acid sequence set forth in SEQID NO:16 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:16. In some embodiments, theintracellular costimulatory domain comprises a CD28 costimulatorydomain, for example, a human CD28 costimulatory domain. In someembodiments, the CD28 costimulatory domain comprises or consists of anamino acid sequence set forth in SEQ ID NO:17 or an amino acid sequencethat is at least 80% identical (e.g., at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical) to the amino acid sequence set forth inSEQ ID NO:17.

In some embodiments, the intracellular signaling domain of the CD22 CARcomprises a CD3 zeta (ζ) signaling domain, for example, a human CD3ζsignaling domain. In some embodiments, the CD3ζ signaling domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:18 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:18.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD22 CAR,including, for example, a CD22 CAR comprising the CD22-specific scFvhaving sequences set forth in SEQ ID NO:45 or SEQ ID NO:54, the CD8αhinge domain of SEQ ID NO:9, the CD8α transmembrane domain of SEQ IDNO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3ζsignaling domain of SEQ ID NO:18, and/or variants (i.e., having asequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD22 CAR,including, for example, a CD22 CAR comprising the CD22-specific scFvhaving sequences set forth in SEQ ID NO:45 or SEQ ID NO:54, the CD28hinge domain of SEQ ID NO:10, the CD8α transmembrane domain of SEQ IDNO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3ζsignaling domain of SEQ ID NO:18, and/or variants (i.e., having asequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD22 CAR,including, for example, a CD22 CAR comprising the CD22-specific scFvhaving sequences set forth in SEQ ID NO:45 or SEQ ID NO:54, the IgG4hinge domain of SEQ ID NO:11 or SEQ ID NO:12, the CD8α transmembranedomain of SEQ ID NO:14, the 4-1BB costimulatory domain of SEQ ID NO:16,the CD3ζ signaling domain of SEQ ID NO:18, and/or variants (i.e., havinga sequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD22 CAR,including, for example, a CD22 CAR comprising the CD22-specific scFvhaving sequences set forth in SEQ ID NO:45 or SEQ ID NO:54, the CD8αhinge domain of SEQ ID NO:9, the CD28 transmembrane domain of SEQ IDNO:15, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3ζsignaling domain of SEQ ID NO:18, and/or variants (i.e., having asequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD22 CAR,including, for example, a CD22 CAR comprising the CD22-specific scFvhaving sequences set forth in SEQ ID NO:45 or SEQ ID NO:54, the CD28hinge domain of SEQ ID NO:10, the CD28 transmembrane domain of SEQ IDNO:15, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3ζsignaling domain of SEQ ID NO:18, and/or variants (i.e., having asequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a CD22 CAR,including, for example, a CD22 CAR comprising the CD22-specific scFvhaving sequences set forth in SEQ ID NO:45 or SEQ ID NO:54, the IgG4hinge domain of SEQ ID NO:11 or SEQ ID NO:12, the CD28 transmembranedomain of SEQ ID NO:15, the 4-1BB costimulatory domain of SEQ ID NO:16,the CD3ζ signaling domain of SEQ ID NO:18, and/or variants (i.e., havinga sequence that is at least 80% identical, for example, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99 identical to the disclosed sequence) thereof.

BCMA CAR

In some embodiments, the CAR is a BCMA CAR (“BCMA-CAR”), and in theseembodiments, the polycistronic vector comprises an expression cassettethat contains a nucleotide sequence encoding a BCMA CAR. BCMA is a tumornecrosis family receptor (TNFR) member expressed on cells of the B celllineage, with the highest expression on terminally differentiated Bcells or mature B lymphocytes. BCMA is involved in mediating thesurvival of plasma cells for maintaining long-term humoral immunity. Theexpression of BCMA has been recently linked to a number of cancers, suchas multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, variousleukemias, and glioblastoma. In some embodiments, the BCMA CAR maycomprise a signal peptide, an extracellular binding domain thatspecifically binds BCMA, a hinge domain, a transmembrane domain, anintracellular costimulatory domain, and/or an intracellular signalingdomain in tandem.

In some embodiments, the signal peptide of the BCMA CAR comprises a CD8αsignal peptide. In some embodiments, the CD8α signal peptide comprisesor consists of an amino acid sequence set forth in SEQ ID NO:6 or anamino acid sequence that is at least 80% identical (e.g., at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in of SEQ ID NO:6. In some embodiments, the signal peptidecomprises an IgK signal peptide. In some embodiments, the IgK signalpeptide comprises or consists of an amino acid sequence set forth in SEQID NO:7 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:7. In some embodiments, thesignal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In someembodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consistsof an amino acid sequence set forth in SEQ ID NO:8 or an amino acidsequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical) to the amino acid sequence setforth in of SEQ ID NO:8.

In some embodiments, the extracellular binding domain of the BCMA CAR isspecific to BCMA, for example, human BCMA. The extracellular bindingdomain of the BCMA CAR can be codon-optimized for expression in a hostcell or to have variant sequences to increase functions of theextracellular binding domain.

In some embodiments, the extracellular binding domain comprises animmunogenically active portion of an immunoglobulin molecule, forexample, an scFv. In some embodiments, the extracellular binding domainof the BCMA CAR is derived from an antibody specific to BCMA, including,for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, andciltacabtagene. In any of these embodiments, the extracellular bindingdomain of the BCMA CAR can comprise or consist of the V_(H), the V_(L),and/or one or more CDRs of any of the antibodies.

In some embodiments, the extracellular binding domain of the BCMA CARcomprises an scFv derived from C11D5.3, a murine monoclonal antibody asdescribed in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013).See also PCT Application Publication No. WO2010/104949. TheC11D5.3-derived scFv may comprise the heavy chain variable region(V_(H)) and the light chain variable region (V_(L)) of C11D5.3 connectedby the Whitlow linker, the amino acid sequences of which is provided inTable 13 below. In some embodiments, the BCMA-specific extracellularbinding domain comprises or consists of an amino acid sequence set forthin SEQ ID NO:63, 64, or 68, or an amino acid sequence that is at least80% identical (e.g., at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical) to the amino acid sequence set forth in of SEQ ID NO:63, 64,or 68. In some embodiments, the BCMA-specific extracellular bindingdomain may comprise one or more CDRs having amino acid sequences setforth in SEQ ID NOs: 65-67 and 69-71. In some embodiments, theBCMA-specific extracellular binding domain may comprise a light chainwith one or more CDRs having amino acid sequences set forth in SEQ IDNOs: 65-67. In some embodiments, the BCMA-specific extracellular bindingdomain may comprise a heavy chain with one or more CDRs having aminoacid sequences set forth in SEQ ID NOs: 69-71. In any of theseembodiments, the BCMA-specific scFv may comprise one or more CDRscomprising one or more amino acid substitutions, or comprising asequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical), to any of the sequencesidentified. In some embodiments, the extracellular binding domain of theBCMA CAR comprises or consists of the one or more CDRs as describedherein.

In some embodiments, the extracellular binding domain of the BCMA CARcomprises an scFv derived from another murine monoclonal antibody,C12A3.2, as described in Carpenter et al., Clin. Cancer Res.19(8):2048-2060 (2013) and PCT Application Publication No.WO2010/104949, the amino acid sequence of which is also provided inTable 13 below. In some embodiments, the BCMA-specific extracellularbinding domain comprises or consists of an amino acid sequence set forthin SEQ ID NO:72, 73, or 77, or an amino acid sequence that is at least80% identical (e.g., at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical) to the amino acid sequence set forth in of SEQ ID NO:72, 73,or 77. In some embodiments, the BCMA-specific extracellular bindingdomain may comprise one or more CDRs having amino acid sequences setforth in SEQ ID NOs: 74-76 and 78-80. In some embodiments, theBCMA-specific extracellular binding domain may comprise a light chainwith one or more CDRs having amino acid sequences set forth in SEQ IDNOs: 74-76. In some embodiments, the BCMA-specific extracellular bindingdomain may comprise a heavy chain with one or more CDRs having aminoacid sequences set forth in SEQ ID NOs: 78-80. In any of theseembodiments, the BCMA-specific scFv may comprise one or more CDRscomprising one or more amino acid substitutions, or comprising asequence that is at least 80% identical (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical), to any of the sequencesidentified. In some embodiments, the extracellular binding domain of theBCMA CAR comprises or consists of the one or more CDRs as describedherein.

In some embodiments, the extracellular binding domain of the BCMA CARcomprises a murine monoclonal antibody with high specificity to humanBCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther.29(5):585-601 (2018)). See also, PCT Application Publication No.WO2012163805.

In some embodiments, the extracellular binding domain of the BCMA CARcomprises single variable fragments of two heavy chains (VHH) that canbind to two epitopes of BCMA as described in Zhao et al., J. Hematol.Oncol. 11(1):141 (2018), also referred to as LCAR-B38M. See also, PCTApplication Publication No. WO2018/028647.

In some embodiments, the extracellular binding domain of the BCMA CARcomprises a fully human heavy-chain variable domain (FHVH) as describedin Lam et al., Nat. Commun. 11(1):283 (2020), also referred to asFHVH33. See also, PCT Application Publication No. WO2019/006072. Theamino acid sequences of FHVH33 and its CDRs are provided in Table 13below. In some embodiments, the BCMA-specific extracellular bindingdomain comprises or consists of an amino acid sequence set forth in SEQID NO:81 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:81. In some embodiments, theBCMA-specific extracellular binding domain may comprise one or more CDRshaving amino acid sequences set forth in SEQ ID NOs: 82-84. In any ofthese embodiments, the BCMA-specific extracellular binding domain maycomprise one or more CDRs comprising one or more amino acidsubstitutions, or comprising a sequence that is at least 80% identical(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical), toany of the sequences identified. In some embodiments, the extracellularbinding domain of the BCMA CAR comprises or consists of the one or moreCDRs as described herein.

In some embodiments, the extracellular binding domain of the BCMA CARcomprises an scFv derived from CT103A (or CAR0085) as described in U.S.Pat. No. 11,026,975 B2, the amino acid sequence of which is provided inTable 13 below. In some embodiments, the BCMA-specific extracellularbinding domain comprises or consists of an amino acid sequence set forthin SEQ ID NO:118, 119, or 123, or an amino acid sequence that is atleast 80% identical (e.g., at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:118, 119, or 123. In some embodiments, the BCMA-specific extracellularbinding domain may comprise one or more CDRs having amino acid sequencesset forth in SEQ ID NOs: 120-122 and 124-126. In some embodiments, theBCMA-specific extracellular binding domain may comprise a light chainwith one or more CDRs having amino acid sequences set forth in SEQ IDNOs: 120-122. In some embodiments, the BCMA-specific extracellularbinding domain may comprise a heavy chain with one or more CDRs havingamino acid sequences set forth in SEQ ID NOs: 124-126. In any of theseembodiments, the BCMA-specific scFv may comprise one or more CDRscomprising one or more amino acid substitutions, or comprising asequence that is at least 8000 identical (e.g., at least 800%, at least8500, at least 900%, at least 9500, at least 960%, at least 97% E atleast 98%, at least 99%, or 100% identical), to any of the sequencesidentified. In some embodiments, the extracellular binding domain of theBCMA CAR comprises or consists of the one or more CDRs as describedherein.

Additionally, CARs and binders directed to BCMA have been described inU. S. Application Publication Nos. 2020/0246381 A1 and 2020/0339699 A1,the entire contents of each of which are incorporated by referenceherein.

TABLE 13 Exemplary sequences of anti-BCMA binder and componentsSEQ ID NO: Amino Acid Sequence Description 63 DIVLTQSPASLAMSLGKRATISCRASAnti-BCMA C11D5.3 ESVSVIGAHLIHWYQQKPGQPPKLLI scFv entire sequence,YLASNLETGVPARFSGSGSGTDFTLT with Whitlow linkerIDPVEEDDVAIYSCLQSRIFPRTFGG GTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASG YTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETS ASTAYLQINNLKYEDTATYFCALDY SYAMDYWGQGTSVTVSS64 DIVLTQSPASLAMSLGKRATISCRAS Anti-BCMA C11D5.3ESVSVIGAHLIHWYQQKPGQPPKLLI scFv light chain variableYLASNLETGVPARFSGSGSGTDFTLT region IDPVEEDDVAIYSCLQSRIFPRTFGG GTKLEIK 65RASESVSVIGAHLIH Anti-BCMA C11D5.3 scFv light chain CDR1 66 LASNLETAnti-BCMA C11D5.3 scFv light chain CDR2 67 LQSRIFPRT Anti-BCMA Cl1D5.3scFv light chain CDR3 68 QIQLVQSGPELKKPGETVKISCKASG Anti-BCMA C11D5.3YTFTDYSINWVKRAPGKGLKWMG scFv heavy chain WINTETREPAYAYDFRGRFAFSLETSvariable region ASTAYLQINNLKYEDTATYFCALDY SYAMDYWGQGTSVTVSS 69 DYSINAnti-BCMA C11D5.3 scFv heavy chain CDR1 70 WINTETREPAYAYDFRGAnti-BCMA C11D5.3 scFv heavy chain CDR2 71 DYSYAMDY Anti-BCMA Cl1D5.3scFv heavy chain CDR3 72 DIVLTQSPPSLAMSLGKRATISCRAS Anti-BCMA C12A3.2ESVTILGSHLIYWYQQKPGQPPTLLI scFv entire sequence,QLASNVQTGVPARFSGSGSRTDFTL with Whitlow linker TIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTK GQIQLVQSGPELKKPGETVKISCKASGYTFRHYSMNWVKQAPGKGLKWM GRINTESGVPIYADDFKGRFAFSVETSASTAYLVINNLKDEDTASYFCSND YLYSLDFWGQGTALTVSS 73DIVLTQSPPSLAMSLGKRATISCRAS Anti-BCMA C12A3.2 ESVTILGSHLIYWYQQKPGQPPTLLIscFv light chain variable QLASNVQTGVPARFSGSGSRTDFTL regionTIDPVEEDDVAVYYCLQSRTIPRTFG GGTKLEIK 74 RASESVTILGSHLIY Anti-BCMA C12A3.2scFv light chain CDR1 75 LASNVQT Anti-BCMA C12A3.2 scFv light chain CDR276 LQSRTIPRT Anti-BCMA C12A3.2 scFv light chain CDR3 77QIQLVQSGPELKKPGETVKISCKASG Anti-BCMA C12A3.2 YTFRHYSMNWVKQAPGKGLKWMGscFv heavy chain RINTESGVPIYADDFKGRFAFSVETS variable regionASTAYLVINNLKDEDTASYFCSNDY LYSLDFWGQGTALTVSS 78 HYSMN Anti-BCMA C12A3.2scFv heavy chain CDR1 79 RINTESGVPIYADDFKG Anti-BCMA C12A3.2scFv heavy chain CDR2 80 DYLYSLDF Anti-BCMA C12A3.2scFv heavy chain CDR3 81 EVQLLESGGGLVQPGGSLRLSCAAS Anti-BCMA FHVH33GFTFSSYAMSWVRQAPGKGLEWVS entire sequence SISGSGDYIYYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAKEG TGANSSLADYRGQGTLVTVSS 82 GFTFSSYAAnti-BCMA FHVH33 CDR1 83 ISGSGDYI Anti-BCMA FHVH33 CDR2 84AKEGTGANSSLADY Anti-BCMA FHVH33 CDR3 118 DIQMTQSPSSLSASVGDRVTITCRASAnti-BCMA CT103A QSISSYLNWYQQKPGKAPKLLIYAA scFv entire sequence,SSLQSGVPSRFSGSGSGTDFTLTISSL with Whitlow linker QPEDFATYYCQQKYDLLTFGGGTKVEIKGSTSGSGKPGSGEGSTKGQLQ LQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSISY SGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRGDTIL DVWGQGTMVTVSS 119 DIQMTQSPSSLSASVGDRVTITCRASAnti-BCMA CT103A QSISSYLNWYQQKPGKAPKLLIYAA scFv light chain variableSSLQSGVPSRFSGSGSGTDFTLTISSL region QPEDFATYYCQQKYDLLTFGGGTK VEIK 120QSISSY Anti-BCMA CT103A scFv light chain CDR1 121 AAS Anti-BCMA CT103AscFv light chain CDR2 122 QQKYDLLT Anti-BCMA CT103AscFv light chain CDR3 123 QLQLQESGPGLVKPSETLSLTCTVSG Anti-BCMA CT103AGSISSSSYYWGWIRQPPGKGLEWIGS scFv heavy chain ISYSGSTYYNPSLKSRVTISVDTSKNvariable region QFSLKLSSVTAADTAVYYCARDRG DTILDVWGQGTMVTVSS 124GGSISSSSYY Anti-BCMA CT103A scFv heavy chain CDR1 125 ISYSGSTAnti-BCMA CT103A scFv heavy chain CDR2 126 ARDRGDTILDV Anti-BCMA CT103AscFv heavy chain CDR3

In some embodiments, the hinge domain of the BCMA CAR comprises a CD8αhinge domain, for example, a human CD8α hinge domain. In someembodiments, the CD8α hinge domain comprises or consists of an aminoacid sequence set forth in SEQ ID NO:9 or an amino acid sequence that isat least 80% identical (e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:9.In some embodiments, the hinge domain comprises a CD28 hinge domain, forexample, a human CD28 hinge domain. In some embodiments, the CD28 hingedomain comprises or consists of an amino acid sequence set forth in SEQID NO:10 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:10. In some embodiments, thehinge domain comprises an IgG4 hinge domain, for example, a human IgG4hinge domain. In some embodiments, the IgG4 hinge domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:11 or SEQ IDNO:12, or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in of SEQ ID NO:11 or SEQ ID NO:12. In someembodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, forexample, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, theIgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acidsequence set forth in SEQ ID NO:13 or an amino acid sequence that is atleast 80% identical (e.g., at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ ID NO:13.

In some embodiments, the transmembrane domain of the BCMA CAR comprisesa CD8α transmembrane domain, for example, a human CD8α transmembranedomain. In some embodiments, the CD8α transmembrane domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:14 or an aminoacid sequence that is at least 80% identical (e.g., at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the amino acid sequenceset forth in SEQ ID NO:14. In some embodiments, the transmembrane domaincomprises a CD28 transmembrane domain, for example, a human CD28transmembrane domain. In some embodiments, the CD28 transmembrane domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:15 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:15.

In some embodiments, the intracellular costimulatory domain of the BCMACAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BBcostimulatory domain. In some embodiments, the 4-1BB costimulatorydomain comprises or consists of an amino acid sequence set forth in SEQID NO:16 or an amino acid sequence that is at least 80% identical (e.g.,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:16. In some embodiments, theintracellular costimulatory domain comprises a CD28 costimulatorydomain, for example, a human CD28 costimulatory domain. In someembodiments, the CD28 costimulatory domain comprises or consists of anamino acid sequence set forth in SEQ ID NO:17 or an amino acid sequencethat is at least 80% identical (e.g., at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical) to the amino acid sequence set forth inSEQ ID NO:17.

In some embodiments, the intracellular signaling domain of the BCMA CARcomprises a CD3 zeta (ζ) signaling domain, for example, a human CD3ζsignaling domain. In some embodiments, the CD3ζ signaling domaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:18 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical) to the aminoacid sequence set forth in SEQ ID NO:18.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a BCMA CAR,including, for example, a BCMA CAR comprising any of the BCMA-specificextracellular binding domains as described, the CD8α hinge domain of SEQID NO:9, the CD8α transmembrane domain of SEQ ID NO:14, the 4-1BBcostimulatory domain of SEQ ID NO:16, the CD3ζ signaling domain of SEQID NO:18, and/or variants (i.e., having a sequence that is at least 80%identical, for example, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99identical to the disclosed sequence) thereof. In any of theseembodiments, the BCMA CAR may additionally comprise a signal peptide(e.g., a CD8α signal peptide) as described.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a BCMA CAR,including, for example, a BCMA CAR comprising any of the BCMA-specificextracellular binding domains as described, the CD8α hinge domain of SEQID NO:9, the CD8α transmembrane domain of SEQ ID NO:14, the CD28costimulatory domain of SEQ ID NO:17, the CD3ζ signaling domain of SEQID NO:18, and/or variants (i.e., having a sequence that is at least 80%identical, for example, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99identical to the disclosed sequence) thereof. In any of theseembodiments, the BCMA CAR may additionally comprise a signal peptide asdescribed.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a BCMA CAR as setforth in SEQ ID NO:127 or is at least 80% identical (e.g., at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical) to the nucleotide sequenceset forth in SEQ ID NO:127 (see Table 14). The encoded BCMA CAR has acorresponding amino acid sequence set forth in SEQ ID NO:128 or is atleast 80% identical (e.g., at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to the amino acid sequence set forth in of SEQ IDNO:128, with the following components: CD8α signal peptide, CT103A scFv(VL-Whitlow linker-VH), CD8α hinge domain, CD8α transmembrane domain,4-1BB costimulatory domain, and CD3ζ signaling domain.

In some embodiments, the polycistronic vector comprises an expressioncassette that contains a nucleotide sequence encoding a commerciallyavailable embodiment of BCMA CAR, including, for example, idecabtagenevicleucel (ide-cel, also called bb2121). In some embodiments, thepolycistronic vector comprises an expression cassette that contains anucleotide sequence encoding idecabtagene vicleucel or portions thereof.Idecabtagene vicleucel comprises a BCMA CAR with the followingcomponents: the BB2121 binder, CD8α hinge domain, CD8α transmembranedomain, 4-11B1 costimulatory domain, and CD3ζ signaling domain.

TABLE 14 Exemplary sequences of BCMA CARs SEQ ID NO: SequenceDescription 127 atggccttaccagtgaccgccttgctcctgccgctggccttgctgcExemplary BCMA tccacgccgccaggccggacatccagatgacccagtctccatcctCAR nucleotide ccctgtctgcatctgtaggagacagagtcaccatcacttgccggg sequencecaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcagcaaaaatacgacctcctcacttttggcggagggaccaaggttgagatcaaaggcagcaccagcggctccggcaagcctggctctggcgagggcagcacaaagggacagctgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccatcagcagtagtagttactactggggctggatccgccagcccccagggaaggggctggagtggattgggagtatctcctatagtgggagcacctactacaacccgtccctcaagagtcgagtcaccatatccgtagacacgtccaagaaccagttctccctgaagctgagttctgtgaccgccgcagacacggcggtgtactactgcgccagagatcgtggagacaccatactagacgtatggggtcagggtacaatggtcaccgtcagctcattcgtgcccgtgttcctgcccgccaaacctaccaccacccctgcccctagacctcccaccccagccccaacaatcgccagccagcctctgtctctgcggcccgaagcctgtagacctgctgccggcggagccgtgcacaccagaggcctggacttcgcctgcgacatctacatctgggcccctctggccggcacctgtggcgtgctgctgctgagcctggtgatcaccctgtactgcaaccaccggaacaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcagatccgccgacgcccctgcctaccagcagggacagaaccagctgtacaacgagctgaacctgggcagacgggaagagtacgacgtgctggacaagcggagaggccgggaccccgagatgggcggaaagcccagacggaagaacccccaggaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcgcggcaagggccacgatggcctgtaccagggcctgagcaccgccaccaaggacacctacgacgccctgcacatgcaggccctgccccccaga 128 MALPVTALLLPLALLLHAARPDIQMTQSPExemplary BCMA SSLSASVGDRVTITCRASQSISSYLNWYQQ CAR amino acidKPGKAPKLLIYAASSLQSGVPSRFSGSGSG sequence TDFTLTISSLQPEDFATYYCQQKYDLLTFGGGTKVEIKGSTSGSGKPGSGEGSTKGQLQ LQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSISYSGSTYYNP SLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRGDTILDVWGQGTMVTVSSFV PVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR

O. Characteristics of Hypoimmunogenic Cells

In some embodiments, the population of hypoimmunogenic stem cellsretains pluripotency as compared to a control stem cell (e.g., awild-type stem cell or immunogenic stem cell). In some embodiments, thepopulation of hypoimmunogenic stem cells retains differentiationpotential as compared to a control stem cell (e.g., a wild-type stemcell or immunogenic stem cell).

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of immune activation in the subject or patient. In some instances,the level of immune activation elicited by the cells is at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lowercompared to the level of immune activation produced by theadministration of immunogenic cells. In some embodiments, theadministered population of hypoimmunogenic cells fails to elicit immuneactivation in the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of T cell response in the subject or patient. In some instances,the level of T cell response elicited by the cells is at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower comparedto the level of T cell response produced by the administration ofimmunogenic cells. In some embodiments, the administered population ofhypoimmunogenic cells fails to elicit a T cell response to the cells inthe subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of NK cell response in the subject or patient. In some instances,the level of NK cell response elicited by the cells is at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower comparedto the level of NK cell response produced by the administration ofimmunogenic cells. In some embodiments, the administered population ofhypoimmunogenic cells fails to elicit an NK cell response to the cellsin the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of macrophage engulfment in the subject or patient. In someinstances, the level of NK cell response elicited by the cells is atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%lower compared to the level of macrophage engulfment produced by theadministration of immunogenic cells. In some embodiments, theadministered population of hypoimmunogenic cells fails to elicitmacrophage engulfment of the cells in the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of systemic TH1 activation in the subject or patient. In someinstances, the level of systemic TH1 activation elicited by the cells isat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%lower compared to the level of systemic TH1 activation produced by theadministration of immunogenic cells. In some embodiments, theadministered population of hypoimmunogenic cells fails to elicitsystemic TH1 activation in the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of NK cell killing in the subject or patient. In some instances,the level of NK cell killing elicited by the cells is at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower comparedto the level of NK cell killing produced by the administration ofimmunogenic cells. In some embodiments, the administered population ofhypoimmunogenic cells fails to elicit NK cell killing in the subject orpatient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of immune activation of peripheral blood mononuclear cells (PBMCs)in the subject or patient. In some instances, the level of immuneactivation of PBMCs elicited by the cells is at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to thelevel of immune activation of PBMCs produced by the administration ofimmunogenic cells. In some embodiments, the administered population ofhypoimmunogenic cells fails to elicit immune activation of PBMCs in thesubject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of donor-specific IgG antibodies in the subject or patient. Insome instances, the level of donor-specific IgG antibodies elicited bythe cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% lower compared to the level of donor-specific IgGantibodies produced by the administration of immunogenic cells. In someembodiments, the administered population of hypoimmunogenic cells failsto elicit donor-specific IgG antibodies in the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of donor-specific IgM antibodies in the subject or patient. Insome instances, the level of donor-specific IgM antibodies elicited bythe cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% lower compared to the level of donor-specific IgMantibodies produced by the administration of immunogenic cells. In someembodiments, the administered population of hypoimmunogenic cells failsto elicit donor-specific IgM antibodies in the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of IgM and IgG antibody production in the subject or patient. Insome instances, the level of IgM and IgG antibody production elicited bythe cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% lower compared to the level of IgM and IgG antibodyproduction produced by the administration of immunogenic cells. In someembodiments, the administered population of hypoimmunogenic cells failsto elicit IgM and IgG antibody production in the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of cytotoxic T cell killing in the subject or patient. In someinstances, the level of cytotoxic T cell killing elicited by the cellsis at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% lower compared to the level of cytotoxic T cell killing produced bythe administration of immunogenic cells. In some embodiments, theadministered population of hypoimmunogenic cells fails to elicitcytotoxic T cell killing in the subject or patient.

In some embodiments, the administered population of hypoimmunogeniccells such as hypoimmunogenic CAR-T cells elicits a decreased or lowerlevel of complement-dependent cytotoxicity (CDC) in the subject orpatient. In some instances, the level of CDC elicited by the cells is atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%lower compared to the level of CDC produced by the administration ofimmunogenic cells. In some embodiments, the administered population ofhypoimmunogenic cells fails to elicit CDC in the subject or patient.

P. Therapeutic Cells from Primary T Cells

Provided herein are hypoimmunogenic cells including, but not limited to,primary T cells that evade immune recognition. In some embodiments, thehypoimmunogenic cells are produced (e.g., generated, cultured, orderived) from T cells such as primary T cells. In some instances,primary T cells are obtained (e.g., harvested, extracted, removed, ortaken) from a subject or an individual. In some embodiments, primary Tcells are produced from a pool of T cells such that the T cells are fromone or more subjects (e.g., one or more human including one or morehealthy humans). In some embodiments, the pool of primary T cells isfrom 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 ormore, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 ormore, or 100 or more subjects. In some embodiments, the donor subject isdifferent from the patient (e.g., the recipient that is administered thetherapeutic cells). In some embodiments, the pool of T cells do notinclude cells from the patient. In some embodiments, one or more of thedonor subjects from which the pool of T cells is obtained are differentfrom the patient.

In some embodiments, the hypoimmunogenic cells do not activate an innateand/or an adaptive immune response in the patient (e.g., recipient uponadministration). Provided are methods of treating a disorder byadministering a population of hypoimmunogenic cells to a subject (e.g.,recipient) or patient in need thereof. In some embodiments, thehypoimmunogenic cells described herein comprise T cells engineered(e.g., are modified) to express a chimeric antigen receptor includingbut not limited to a chimeric antigen receptor described herein. In someinstances, the T cells are populations or subpopulations of primary Tcells from one or more individuals. In some embodiments, the T cellsdescribed herein such as the engineered or modified T cells comprisereduced expression of an endogenous T cell receptor.

In some embodiments, the present disclosure is directed tohypoimmunogenic primary T cells that overexpress CD47 and CARs, and havereduced expression or lack expression of MHC class I and/or MHC class IIhuman leukocyte antigens and have reduced expression or lack expressionof TCR complex molecules. The cells outlined herein overexpress CD47 andCARs and evade immune recognition. In some embodiments, the primary Tcells display reduced levels or activity of MHC class I antigens, MHCclass II antigens, and/or TCR complex molecules. In certain embodiments,primary T cells overexpress CD47 and CARs and harbor a genomicmodification in the B2M gene. In some embodiments, T cells overexpressCD47 and CARs and harbor a genomic modification in the CIITA gene. Insome embodiments, primary T cells overexpress CD47 and CARs and harbor agenomic modification in the TRAC gene. In some embodiments, primary Tcells overexpress CD47 and CARs and harbor a genomic modification in theTRB gene. In some embodiments, T cells overexpress CD47 and CARs andharbor genomic modifications in one or more of the following genes: theB2M, CIITA, TRAC and TRB genes.

Exemplary T cells of the present disclosure are selected from the groupconsisting of cytotoxic T cells, helper T cells, memory T cells, centralmemory T cells, effector memory T cells, effector memory RA T cells,regulatory T cells, tissue infiltrating lymphocytes, and combinationsthereof. In certain embodiments, the T cells express CCR7, CD27, CD28,and CD45RA. In some embodiments, the central T cells express CCR7, CD27,CD28, and CD45RO. In other embodiments, the effector memory T cellsexpress PD-1, CD27, CD28, and CD45RO. In other embodiments, the effectormemory RA T cells express PD-1, CD57, and CD45RA.

In some embodiments, the T cell is a modified (e.g., an engineered) Tcell. In some cases, the modified T cell comprise a modification causingthe cell to express at least one chimeric antigen receptor thatspecifically binds to an antigen or epitope of interest expressed on thesurface of at least one of a damaged cell, a dysplastic cell, aninfected cell, an immunogenic cell, an inflamed cell, a malignant cell,a metaplastic cell, a mutant cell, and combinations thereof. In othercases, the modified T cell comprise a modification causing the cell toexpress at least one protein that modulates a biological effect ofinterest in an adjacent cell, tissue, or organ when the cell is inproximity to the adjacent cell, tissue, or organ. Useful modificationsto primary T cells are described in detail in US2016/0348073 andWO2020/018620, the disclosures of which are incorporated herein in theirentireties.

In some embodiments, the hypoimmunogenic cells described herein compriseT cells that are engineered (e.g., are modified) to express a chimericantigen receptor including but not limited to a chimeric antigenreceptor described herein. In some instances, the T cells arepopulations or subpopulations of primary T cells from one or moreindividuals. In some embodiments, the T cells described herein such asthe engineered or modified T cells include reduced expression of anendogenous T cell receptor. In some embodiments, the T cells describedherein such as the engineered or modified T cells include reducedexpression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Inother embodiments, the T cells described herein such as the engineeredor modified T cells include reduced expression of programmed cell death(PD-1). In certain embodiments, the T cells described herein such as theengineered or modified T cells include reduced expression of CTLA-4 andPD-1. Methods of reducing or eliminating expression of CTLA-4, PD-1 andboth CTLA-4 and PD-1 can include any recognized by those skilled in theart, such as but not limited to, genetic modification technologies thatutilize rare-cutting endonucleases and RNA silencing or RNA interferencetechnologies. Non-limiting examples of a rare-cutting endonucleaseinclude any Cas protein, TALEN, zinc finger nuclease, meganuclease, andhoming endonuclease. In some embodiments, an exogenous nucleic acidencoding a polypeptide as disclosed herein (e.g., a chimeric antigenreceptor, CD47, or another tolerogenic factor disclosed herein) isinserted at a CTLA-4 and/or PD-1 gene locus. In some embodiments, theexogenous polynucleotide is inserted into at least one allele of thecell using viral transduction, for example, with a vector. In someembodiments, the vector is a pseudotyped, self-inactivating lentiviralvector that carries the exogenous polynucleotide. In some embodiments,the vector is a self-inactivating lentiviral vector pseudotyped with avesicular stomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

In some embodiments, the T cells described herein such as the engineeredor modified T cells include enhanced expression of PD-L1.

In some embodiments, the hypoimmunogenic T cell includes apolynucleotide encoding a CAR, wherein the polynucleotide is inserted ina genomic locus. In some embodiments, the polynucleotide encoding theCAR is randomly integrated into the genome of the cell. In someembodiments, the polynucleotide encoding the CAR is randomly integratedinto the genome of the cell via viral vector transduction. In someembodiments, the polynucleotide encoding the CAR is randomly integratedinto the genome of the cell via lentiviral vector transduction. In someembodiments, the polynucleotide is inserted into a safe harbor or targetlocus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26,SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91),HMGB1, ABO, RHD, FUT1, or KDM5D gene locus. In some embodiments, thepolynucleotide is inserted in a B2M, CIITA, TRAC, TRB, PD-1 or CTLA-4gene.

In some embodiments, the hypoimmunogenic T cell includes apolynucleotide encoding a CAR that is expressed in a cell using anexpression vector. In some embodiments, the CAR is introduced to thecell using a viral expression vector that mediates integration of theCAR sequence into the genome of the cell. For example, the expressionvector for expressing the CAR in a cell comprises a polynucleotidesequence encoding the CAR. The expression vector can be an inducibleexpression vector. The expression vector can be a viral vector, such asbut not limited to, a lentiviral vector.

Hypoimmunogenic T cells provided herein are useful for the treatment ofsuitable cancers including, but not limited to, B cell acutelymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, livercancer, pancreatic cancer, breast cancer, ovarian cancer, colorectalcancer, lung cancer, non-small cell lung cancer, acute myeloid lymphoidleukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma,pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamouscell carcinoma, hepatocellular carcinoma, and bladder cancer.

Q. Therapeutic Cells Differentiated from Hypoimmunogenic PluripotentStem Cells

Provided herein are hypoimmunogenic cells including, cells derived frompluripotent stem cells, that evade immune recognition. In someembodiments, the cells do not activate an innate and/or an adaptiveimmune response in the patient or subject (e.g., recipient uponadministration). Provided are methods of treating a disorder comprisingrepeat dosing of a population of hypoimmunogenic cells to a recipientsubject in need thereof.

In some embodiments, the pluripotent stem cell and any celldifferentiated from such a pluripotent stem cell is modified to exhibitreduced expression of MHC class I human leukocyte antigens. In otherembodiments, the pluripotent stem cell and any cell differentiated fromsuch a pluripotent stem cell is modified to exhibit reduced expressionof MHC class II human leukocyte antigens. In certain embodiments, thepluripotent stem cell and any cell differentiated from such apluripotent stem cell is modified to exhibit reduced expression of TCRcomplexes. In some embodiments, the pluripotent stem cell and any celldifferentiated from such a pluripotent stem cell is modified to exhibitreduced expression of MHC class I and II human leukocyte antigens. Insome embodiments, the pluripotent stem cell and any cell differentiatedfrom such a pluripotent stem cell is modified to exhibit reducedexpression of MHC class I and II human leukocyte antigens and TCRcomplexes.

In some embodiments, the pluripotent stem cell and any celldifferentiated from such a pluripotent stem cell is modified to exhibitreduced expression of MHC class I and/or II human leukocyte antigens andexhibit increased CD47 expression. In some instances, the celloverexpresses CD47 by harboring one or more CD47 transgenes. In someembodiments, the pluripotent stem cell and any cell differentiated fromsuch a pluripotent stem cell is modified to exhibit reduced expressionof MHC class I and II human leukocyte antigens and exhibit increasedCD47 expression. In some embodiments, the pluripotent stem cell and anycell differentiated from such a pluripotent stem cell is modified toexhibit reduced expression of MHC class I and II human leukocyteantigens and TCR complexes and exhibit increased CD47 expression.

In some embodiments, the pluripotent stem cell and any celldifferentiated from such a pluripotent stem cell is modified to exhibitreduced expression of MHC class I and/or II human leukocyte antigens, toexhibit increased CD47 expression, and to exogenously express a chimericantigen receptor. In some instances, the cell overexpresses CD47polypeptides by harboring one or more CD47 transgenes. In someinstances, the cell overexpresses CAR polypeptides by harboring one ormore CAR transgenes. In some embodiments, the pluripotent stem cell andany cell differentiated from such a pluripotent stem cell is modified toexhibit reduced expression of MHC class I and II human leukocyteantigens, exhibit increased CD47 expression, and to exogenously expressa chimeric antigen receptor. In some embodiments, the pluripotent stemcell and any cell differentiated from such a pluripotent stem cell ismodified to exhibit reduced expression of MHC class I and II humanleukocyte antigens and TCR complexes, to exhibit increased CD47expression, and to exogenously express a chimeric antigen receptor.

Such pluripotent stem cells are hypoimmunogenic stem cells. Suchdifferentiated cells are hypoimmunogenic cells.

Any of the pluripotent stem cells described herein can be differentiatedinto any cells of an organism and tissue. In some embodiments, the cellsexhibit reduced expression of MHC class I and/or II human leukocyteantigens and reduced expression of TCR complexes. In some instances,expression of MHC class I and/or II human leukocyte antigens is reducedcompared to unmodified or wild-type cell of the same cell type. In someinstances, expression of TCR complexes is reduced compared to unmodifiedor wild-type cell of the same cell type. In some embodiments, the cellsexhibit increased CD47 expression. In some instances, expression of CD47is increased in cells encompassed by the present disclosure as comparedto unmodified or wild-type cells of the same cell type. In someembodiments, the cells exhibit exogenous CAR expression. Methods forreducing levels of MHC class I and/or II human leukocyte antigens andTCR complexes and increasing the expression of CD47 and CARs aredescribed herein.

In some embodiments, the cells used in the methods described hereinevade immune recognition and responses when administered to a patient(e.g., recipient subject). The cells can evade killing by immune cellsin vitro and in vivo. In some embodiments, the cells evade killing bymacrophages and NK cells. In some embodiments, the cells are ignored byimmune cells or a subject's immune system. In other words, the cellsadministered in accordance with the methods described herein are notdetectable by immune cells of the immune system. In some embodiments,the cells are cloaked and therefore avoid immune rejection.

Methods of determining whether a pluripotent stem cell and any celldifferentiated from such a pluripotent stem cell evades immunerecognition include, but are not limited to, IFN-7 Elispot assays,microglia killing assays, cell engraftment animal models, cytokinerelease assays, ELISAs, killing assays using bioluminescence imaging orchromium release assay or a real-time, quantitative microelectronicbiosensor system for cell analysis (xCELLigence© RTCA system, Agilent),mixed-lymphocyte reactions, immunofluorescence analysis, etc.

Therapeutic cells outlined herein are useful to treat a disorder suchas, but not limited to, a cancer, a genetic disorder, a chronicinfectious disease, an autoimmune disorder, a neurological disorder, andthe like.

1. T Lymphocytes Differentiated from Hypoimmunogenic Pluripotent Cells

Provided herein, T lymphocytes (T cells, including primary T cells) arederived from the HIP cells described herein (e.g., hypoimmunogeniciPSCs). Methods for generating T cells, including CAR-T cells, frompluripotent stem cells (e.g., iPSCs) are described, for example, inIriguchi et al., Nature Communications 12, 430 (2021); Themeli et al.,Cell Stem Cell, 16(4):357-366 (2015); Themeli et al., NatureBiotechnology 31:928-933 (2013).

T lymphocyte derived hypoimmunogenic cells include, but are not limitedto, primary T cells that evade immune recognition. In some embodiments,the hypoimmunogenic cells are produced (e.g., generated, cultured, orderived) from T cells such as primary T cells. In some instances,primary T cells are obtained (e.g., harvested, extracted, removed, ortaken) from a subject or an individual. In some embodiments, primary Tcells are produced from a pool of T cells such that the T cells are fromone or more subjects (e.g., one or more human including one or morehealthy humans). In some embodiments, the pool of primary T cells isfrom 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 ormore, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 ormore, or 100 or more subjects. In some embodiments, the donor subject isdifferent from the patient (e.g., the recipient that is administered thetherapeutic cells). In some embodiments, the pool of T cells does notinclude cells from the patient. In some embodiments, one or more of thedonor subjects from which the pool of T cells is obtained are differentfrom the patient.

In some embodiments, the hypoimmunogenic cells do not activate an immuneresponse in the patient (e.g., recipient upon administration). Providedare methods of treating a disorder by administering a population ofhypoimmunogenic cells to a subject (e.g., recipient) or patient in needthereof. In some embodiments, the hypoimmunogenic cells described hereincomprise T cells engineered (e.g., are modified) to express a chimericantigen receptor including but not limited to a chimeric antigenreceptor described herein. In some instances, the T cells arepopulations or subpopulations of primary T cells from one or moreindividuals. In some embodiments, the T cells described herein such asthe engineered or modified T cells comprise reduced expression of anendogenous T cell receptor.

In some embodiments, the HIP-derived T cell includes a chimeric antigenreceptor (CAR). Any suitable CAR can be included in the hyHIP-derived Tcell, including the CARs described herein. In some embodiments, thehypoimmunogenic induced pluripotent stem cell-derived T cell includes apolynucleotide encoding a CAR, wherein the polynucleotide is inserted ina genomic locus. In some embodiments, the polynucleotide is insertedinto a safe harbor or target locus. In some embodiments, thepolynucleotide is inserted in a B2M, CIITA, TRAC, TRB, PD-1 or CTLA-4gene. Any suitable method can be used to insert the CAR into the genomiclocus of the hypoimmunogenic cell including the gene editing methodsdescribed herein (e.g., a CRISPR/Cas system).

HIP-derived T cells provided herein are useful for the treatment ofsuitable cancers including, but not limited to, B cell acutelymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, livercancer, pancreatic cancer, breast cancer, ovarian cancer, colorectalcancer, lung cancer, non-small cell lung cancer, acute myeloid lymphoidleukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma,pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamouscell carcinoma, hepatocellular carcinoma, and bladder cancer.

2. NK Cells Derived from Hypoimmunogenic Pluripotent Cells

Provided herein, natural killer (NK) cells are derived from the HIPcells described herein (e.g., hypoimmunogenic iPSCs).

NK cells (also defined as ‘large granular lymphocytes’) represent a celllineage differentiated from the common lymphoid progenitor (which alsogives rise to B lymphocytes and T lymphocytes). Unlike T-cells, NK cellsdo not naturally comprise CD3 at the plasma membrane. Importantly, NKcells do not express a TCR and typically also lack otherantigen-specific cell surface receptors (as well as TCRs and CD3, theyalso do not express immunoglobulin B-cell receptors, and insteadtypically express CD16 and CD56). NK cell cytotoxic activity does notrequire sensitization but is enhanced by activation with a variety ofcytokines including IL-2. NK cells are generally thought to lackappropriate or complete signaling pathways necessary forantigen-receptor-mediated signaling, and thus are not thought to becapable of antigen receptor-dependent signaling, activation andexpansion. NK cells are cytotoxic, and balance activating and inhibitoryreceptor signaling to modulate their cytotoxic activity. For instance,NK cells expressing CD16 may bind to the Fc domain of antibodies boundto an infected cell, resulting in NK cell activation. By contrast,activity is reduced against cells expressing high levels of MHC class Iproteins. On contact with a target cell NK cells release proteins suchas perforin, and enzymes such as proteases (granzymes). Perforin canform pores in the cell membrane of a target cell, inducing apoptosis orcell lysis.

There are a number of techniques that can be used to generate NK cells,including CAR-NK-cells, from pluripotent stem cells (e.g., iPSC); see,for example, Zhu et al., Methods Mol Biol. 2019; 2048:107-119; Knorr etal., Stem Cells Transl Med. 2013 2(4):274-83. doi:10.5966/sctm.2012-0084; Zeng et al., Stem Cell Reports. 2017 Dec. 12;9(6):1796-1812; Ni et al., Methods Mol Biol. 2013; 1029:33-41;Bernareggi et al., Exp Hematol. 2019 71:13-23; Shankar et al., Stem CellRes Ther. 2020; 11(1):234, all of which are incorporated herein byreference in their entirety and specifically for the methodologies andreagents for differentiation. Differentiation can be assayed as is knownin the art, generally by evaluating the presence of NK cell associatedand/or specific markers, including, but not limited to, CD56, KIRs,CD16, NKp44, NKp46, NKG2D, TRAIL, CD122, CD27, CD244, NK1.1, NKG2A/C,NCR1, Ly49, CD49b, CD11b, KLRG1, CD43, CD62L, and/or CD226.

In some embodiments, the hypoimmunogenic pluripotent cells aredifferentiated into hepatocytes to address loss of the hepatocytefunctioning or cirrhosis of the liver. There are a number of techniquesthat can be used to differentiate HIP cells into hepatocytes; see forexample, Pettinato et al., doi: 10.1038/spre32888, Snykers et al.,Methods Mol Biol., 2011 698:305-314, Si-Tayeb et al., Hepatology, 2010,51:297-305 and Asgari et al., Stem Cell Rev., 2013, 9(4):493-504, all ofwhich are incorporated herein by reference in their entirety andspecifically for the methodologies and reagents for differentiation.Differentiation can be assayed as is known in the art, generally byevaluating the presence of hepatocyte associated and/or specificmarkers, including, but not limited to, albumin, alpha fetoprotein, andfibrinogen. Differentiation can also be measured functionally, such asthe metabolization of ammonia, LDL storage and uptake, ICG uptake andrelease, and glycogen storage.

In some embodiments, the NK cells do not activate an innate and/or anadaptive immune response in the patient (e.g., recipient uponadministration). Provided are methods of treating a disorder byadministering a population of NK cells to a subject (e.g., recipient) orpatient in need thereof. In some embodiments, the NK cells describedherein comprise NK cells engineered (e.g., are modified) to express achimeric antigen receptor including but not limited to a chimericantigen receptor described herein. Any suitable CAR can be included inthe NK cells, including the CARs described herein. In some embodiments,the NK cell includes a polynucleotide encoding a CAR, wherein thepolynucleotide is inserted in a genomic locus. In some embodiments, thepolynucleotide is inserted into a safe harbor or a target locus. In someembodiments, the polynucleotide is inserted in a B2M, CIITA, PD1 orCTLA4 gene. Any suitable method can be used to insert the CAR into thegenomic locus of the NK cell including the gene editing methodsdescribed herein (e.g., a CRISPR/Cas system).

R. Methods of Genetic Modifications

In some embodiments, a vector herein is a nucleic acid molecule capabletransferring or transporting another nucleic acid molecule, includinginto the cell or into genome of a cell. The transferred nucleic acid isgenerally linked to, e.g., inserted into, the vector nucleic acidmolecule. A vector may include sequences that direct autonomousreplication in a cell or may include sequences sufficient to allowintegration into host cell DNA. Useful vectors include, for example,plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids,bacterial artificial chromosomes, and viral vectors. Useful viralvectors include, e.g., replication defective retroviruses andlentiviruses. Non-viral vectors may require a delivery vehicle tofacilitate entry of the nucleic acid molecule into a cell.

A viral vector can comprise a nucleic acid molecule that includesvirus-derived nucleic acid elements that typically facilitate transferof the nucleic acid molecule or integration into the genome of a cell orto a viral particle that mediates nucleic acid transfer. Viral particleswill typically include various viral components and sometimes also hostcell components in addition to nucleic acid(s). A viral vector cancomprise, e.g., a virus or viral particle capable of transferring anucleic acid into a cell, or to the transferred nucleic acid (e.g., asnaked DNA). Viral vectors and transfer plasmids can comprise structuraland/or functional genetic elements that are primarily derived from avirus. A retroviral vector can comprise a viral vector or plasmidcontaining structural and functional genetic elements, or portionsthereof, that are primarily derived from a retrovirus.

In some vectors described herein, at least part of one or more proteincoding regions that contribute to or are essential for replication maybe absent compared to the corresponding wild-type virus. This makes theviral vector replication-defective. In some embodiments, the vector iscapable of transducing a target non-dividing host cell and/orintegrating its genome into a host genome.

In some embodiments, the retroviral nucleic acid comprises one or moreof (e.g., all of): a 5′ promoter (e.g., to control expression of theentire packaged RNA), a 5′ LTR (e.g., that includes R (polyadenylationtail signal) and/or U5 which includes a primer activation signal), aprimer binding site, a psi packaging signal, a RRE element for nuclearexport, a promoter directly upstream of the transgene to controltransgene expression, a transgene (or other exogenous agent element), apolypurine tract, and a 3′ LTR (e.g., that includes a mutated U3, a R,and U5). In some embodiments, the retroviral nucleic acid furthercomprises one or more of a cPPT, a WPRE, and/or an insulator element.

A retrovirus typically replicates by reverse transcription of itsgenomic RNA into a linear double-stranded DNA copy and subsequentlycovalently integrates its genomic DNA into a host genome. The structureof a wild-type retrovirus genome often comprises a 5′ long terminalrepeat (LTR) and a 3′ LTR, between or within which are located apackaging signal to enable the genome to be packaged, a primer bindingsite, integration sites to enable integration into a host cell genomeand gag, pol and env genes encoding the packaging components whichpromote the assembly of viral particles. More complex retroviruses haveadditional features, such as rev and RRE sequences in HIV, which enablethe efficient export of RNA transcripts of the integrated provirus fromthe nucleus to the cytoplasm of an infected target cell. In theprovirus, the viral genes are flanked at both ends by regions calledlong terminal repeats (LTRs). The LTRs are involved in proviralintegration and transcription. LTRs also serve as enhancer-promotersequences and can control the expression of the viral genes.Encapsidation of the retroviral RNAs occurs by virtue of a psi sequencelocated at the 5′ end of the viral genome.

The LTRs themselves are typically similar (e.g., identical) sequencesthat can be divided into three elements, which are called U3, R and U5.U3 is derived from the sequence unique to the 3′ end of the RNA. R isderived from a sequence repeated at both ends of the RNA and U5 isderived from the sequence unique to the 5′ end of the RNA. The sizes ofthe three elements can vary considerably among different retroviruses.

For the viral genome, the site of transcription initiation is typicallyat the boundary between U3 and R in one LTR and the site of poly (A)addition (termination) is at the boundary between R and U5 in the otherLTR. U3 contains most of the transcriptional control elements of theprovirus, which include the promoter and multiple enhancer sequencesresponsive to cellular and in some cases, viral transcriptionalactivator proteins. Some retroviruses comprise any one or more of thefollowing genes that code for proteins that are involved in theregulation of gene expression: tot, rev, tax and rex.

With regard to the structural genes gag, pol and env themselves, gagencodes the internal structural protein of the virus. Gag protein isproteolytically processed into the mature proteins MA (matrix), CA(capsid) and NC (nucleocapsid). The pol gene encodes the reversetranscriptase (RT), which contains DNA polymerase, associated RNase Hand integrase (IN), which mediate replication of the genome. The envgene encodes the surface (SU) glycoprotein and the transmembrane (TM)protein of the virion, which form a complex that interacts specificallywith cellular receptor proteins. This interaction promotes infection,e.g., by fusion of the viral membrane with the cell membrane.

In a replication-defective retroviral vector genome gag, pol and env maybe absent or not functional. The R regions at both ends of the RNA aretypically repeated sequences. U5 and U3 represent unique sequences atthe 5′ and 3′ ends of the RNA genome respectively. Retroviruses may alsocontain additional genes which code for proteins other than gag, pol andenv. Examples of additional genes include (in HIV), one or more of vif,vpr, vpx, vpu, tat, rev and nef. EIAV has (amongst others) theadditional gene S2.

Illustrative retroviruses suitable for use in particular embodiments,include, but are not limited to: Moloney murine leukemia virus (M-MuLV),Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus(GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemiavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) andlentivirus.

In some embodiments the retrovirus is a Gammretrovirus. In someembodiments the retrovirus is an Epsilonretrovirus. In some embodimentsthe retrovirus is an Alpharetrovirus. In some embodiments the retrovirusis a Betaretro virus. In some embodiments the retrovirus is a Deltaretrovirus. In some embodiments the retrovirus is a Spumaretrovirus. In someembodiments the retrovirus is an endogenous retrovirus. In someembodiments the retrovirus is a lentivirus.

In some embodiments, a retroviral or lentivirus vector further comprisesone or more insulator elements, e.g., an insulator element describedherein. In various embodiments, the vectors comprise a promoter operablylinked to a polynucleotide encoding an exogenous agent. The vectors mayhave one or more LTRs, wherein either LTR comprises one or moremodifications, such as one or more nucleotide substitutions, additions,or deletions. The vectors may further comprise one of more accessoryelements to increase transduction efficiency (e.g., a cPPT/FLAP), viralpackaging (e.g., a Psi (Y) packaging signal, RRE), and/or other elementsthat increase exogenous gene expression (e.g., poly (A) sequences), andmay optionally comprise a WPRE or HPRE. In some embodiments, alentiviral nucleic acid comprises one or more of, e.g., all of, e.g.,from 5′ to 3′, a promoter (e.g., CMV), an R sequence (e.g., comprisingTAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., forreverse transcription), a DIS sequence (e.g., for genome dimerization),a psi packaging signal, a partial gag sequence, an RRE sequence (e.g.,for nuclear export), a cPPT sequence (e.g., for nuclear import), apromoter to drive expression of the exogenous agent, a gene encoding theexogenous agent, a WPRE sequence (e.g., for efficient transgeneexpression), a PPT sequence (e.g., for reverse transcription), an Rsequence (e.g., for polyadenylation and termination), and a U5 signal(e.g., for integration).

Illustrative lentiviruses include, but are not limited to: HIV (humanimmunodeficiency virus; including HIV type 1, and HIV type 2);visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus(CAEV); equine infectious anemia virus (EIAV); feline immunodeficiencyvirus (FIV); bovine immune deficiency virus (BIV); and simianimmunodeficiency virus (SIV). In some embodiments, HIV based vectorbackbones (i.e., HIV cis-acting sequence elements) are used. Alentivirus vector can comprise a viral vector or plasmid containingstructural and functional genetic elements, or portions thereof,including LTRs that are primarily derived from a lentivirus.

In embodiments, a lentivirus vector (e.g., lentiviral expression vector)may comprise a lentiviral transfer plasmid (e.g., as naked DNA) or aninfectious lentiviral particle. With respect to elements such as cloningsites, promoters, regulatory elements, heterologous nucleic acids, etc.,it is to be understood that the sequences of these elements can bepresent in RNA form in lentiviral particles and can be present in DNAform in DNA plasmids.

In embodiments, a lentivirus vector is a vector with sufficientretroviral genetic information to allow packaging of an RNA genome, inthe presence of packaging components, into a viral particle capable ofinfecting a target cell. Infection of the target cell can comprisereverse transcription and integration into the target cell genome. TheRLV typically carries non-viral coding sequences which are to bedelivered by the vector to the target cell. In embodiments, an RLV isincapable of independent replication to produce infectious retroviralparticles within the target cell. Usually the RLV lacks a functionalgag-pol and/or env gene and/or other genes involved in replication. Thevector may be configured as a split-intron vector, e.g., as described inPCT patent application WO 99/15683, which is herein incorporated byreference in its entirety.

In some embodiments, the lentivirus vector comprises a minimal viralgenome, e.g., the viral vector has been manipulated so as to remove thenon-essential elements and to retain the essential elements in order toprovide the required functionality to infect, transduce and deliver anucleotide sequence of interest to a target host cell, e.g., asdescribed in WO 98/17815, which is herein incorporated by reference inits entirety.

A minimal lentiviral genome may comprise, e.g., (5′)R-U5-one or morefirst nucleotide sequences-U3-R(3′) However, the plasmid vector used toproduce the lentiviral genome within a source cell can also includetranscriptional regulatory control sequences operably linked to thelentiviral genome to direct transcription of the genome in a sourcecell. These regulatory sequences may comprise the natural sequencesassociated with the transcribed retroviral sequence, e.g., the 5′ U3region, or they may comprise a heterologous promoter such as anotherviral promoter, for example the CMV promoter. Some lentiviral genomescomprise additional sequences to promote efficient virus production. Forexample, in the case of HIV, rev and RRE sequences may be included.

In some embodiments, the rare-cutting endonuclease is introduced into acell containing the target polynucleotide sequence in the form of anucleic acid encoding a rare-cutting endonuclease. The process ofintroducing the nucleic acids into cells can be achieved by any suitabletechnique. Suitable techniques include calcium phosphate orlipid-mediated transfection, electroporation, and transduction orinfection using a viral vector. In some embodiments, the nucleic acidcomprises DNA. In some embodiments, the nucleic acid comprises amodified DNA, as described herein. In some embodiments, the nucleic acidcomprises mRNA. In some embodiments, the nucleic acid comprises amodified mRNA, as described herein (e.g., a synthetic, modified mRNA).

The present disclosure contemplates altering target polynucleotidesequences in any manner which is available to the skilled artisanutilizing a gene editing system (e.g., CRISPR/Cas) of the presentdisclosure. Any CRISPR/Cas system that is capable of altering a targetpolynucleotide sequence in a cell can be used. Such CRISPR-Cas systemscan employ a variety of Cas proteins (Haft et al. PLoS Comput Biol.2005; 1(6)e60). The molecular machinery of such Cas proteins that allowsthe CRISPR/Cas system to alter target polynucleotide sequences in cellsinclude RNA binding proteins, endo- and exo-nucleases, helicases, andpolymerases. In some embodiments, the CRISPR/Cas system is a CRISPR typeI system. In some embodiments, the CRISPR/Cas system is a CRISPR type IIsystem. In some embodiments, the CRISPR/Cas system is a CRISPR type Vsystem.

The CRISPR/Cas systems of the present disclosure can be used to alterany target polynucleotide sequence in a cell. Those skilled in the artwill readily appreciate that desirable target polynucleotide sequencesto be altered in any particular cell may correspond to any genomicsequence for which expression of the genomic sequence is associated witha disorder or otherwise facilitates entry of a pathogen into the cell.For example, a desirable target polynucleotide sequence to alter in acell may be a polynucleotide sequence corresponding to a genomicsequence which contains a disease associated single polynucleotidepolymorphism. In such example, the CRISPR/Cas systems of the presentdisclosure can be used to correct the disease associated SNP in a cellby replacing it with a wild-type allele. As another example, apolynucleotide sequence of a target gene which is responsible for entryor proliferation of a pathogen into a cell may be a suitable target fordeletion or insertion to disrupt the function of the target gene toprevent the pathogen from entering the cell or proliferating inside thecell.

In some embodiments, the target polynucleotide sequence is a genomicsequence. In some embodiments, the target polynucleotide sequence is ahuman genomic sequence. In some embodiments, the target polynucleotidesequence is a mammalian genomic sequence. In some embodiments, thetarget polynucleotide sequence is a vertebrate genomic sequence.

In some embodiments, a CRISPR/Cas system of the present disclosureincludes a Cas protein and at least one to two ribonucleic acids thatare capable of directing the Cas protein to and hybridizing to a targetmotif of a target polynucleotide sequence. As used herein, “protein” and“polypeptide” are used interchangeably to refer to a series of aminoacid residues joined by peptide bonds (i.e., a polymer of amino acids)and include modified amino acids (e.g., phosphorylated, glycated,glycosylated, etc.) and amino acid analogs. Exemplary polypeptides orproteins include gene products, naturally occurring proteins, homologs,paralogs, fragments and other equivalents, variants, and analogs of theabove.

In some embodiments, a Cas protein comprises one or more amino acidsubstitutions or modifications. In some embodiments, the one or moreamino acid substitutions comprises a conservative amino acidsubstitution. In some instances, substitutions and/or modifications canprevent or reduce proteolytic degradation and/or extend the half-life ofthe polypeptide in a cell. In some embodiments, the Cas protein cancomprise a peptide bond replacement (e.g., urea, thiourea, carbamate,sulfonyl urea, etc.). In some embodiments, the Cas protein can comprisea naturally occurring amino acid. In some embodiments, the Cas proteincan comprise an alternative amino acid (e.g., D-amino acids, beta-aminoacids, homocysteine, phosphoserine, etc.). In some embodiments, a Casprotein can comprise a modification to include a moiety (e.g.,PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

In some embodiments, a Cas protein comprises a core Cas protein, isoformthereof, or any Cas-like protein with similar function or activity ofany Cas protein or isoform thereof. In some embodiments, a Cas proteincomprises a core Cas protein. Exemplary Cas core proteins include, butare not limited to Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 andCas9. In some embodiments, a Cas protein comprises type V Cas protein.In some embodiments, a Cas protein comprises a Cas protein of an E. colisubtype (also known as CASS2). Exemplary Cas proteins of the E. Colisubtype include, but are not limited to Cse1, Cse2, Cse3, Cse4, andCas5e. In some embodiments, a Cas protein comprises a Cas protein of theYpest subtype (also known as CASS3). Exemplary Cas proteins of the Ypestsubtype include, but are not limited to Csy1, Csy2, Csy3, and Csy4. Insome embodiments, a Cas protein comprises a Cas protein of the Nmenisubtype (also known as CASS4). Exemplary Cas proteins of the Nmenisubtype include, but are not limited to Csn1 and Csn2. In someembodiments, a Cas protein comprises a Cas protein of the Dvulg subtype(also known as CASS1). Exemplary Cas proteins of the Dvulg subtypeinclude Csd1, Csd2, and Cas5d. In some embodiments, a Cas proteincomprises a Cas protein of the Tneap subtype (also known as CASS7).Exemplary Cas proteins of the Tneap subtype include, but are not limitedto, Cst1, Cst2, Cas5t. In some embodiments, a Cas protein comprises aCas protein of the Hmari subtype. Exemplary Cas proteins of the Hmarisubtype include, but are not limited to Csh1, Csh2, and Cas5h. In someembodiments, a Cas protein comprises a Cas protein of the Apern subtype(also known as CASS5). Exemplary Cas proteins of the Apern subtypeinclude, but are not limited to Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a.In some embodiments, a Cas protein comprises a Cas protein of the Mtubesubtype (also known as CASS6). Exemplary Cas proteins of the Mtubesubtype include, but are not limited to Csm1, Csm2, Csm3, Csm4, andCsm5. In some embodiments, a Cas protein comprises a RAMP module Casprotein. Exemplary RAMP module Cas proteins include, but are not limitedto, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6. See, e.g., Klompe et al.,Nature 571, 219-225 (2019); Strecker et al., Science 365, 48-53 (2019).Examples of Cas proteins include, but are not limited to: Cas3, Cas8a,Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and/orGSU0054. In some embodiments, a Cas protein comprises Cas3, Cas8a, Cas5,Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and/or GSU0054.Examples of Cas proteins include, but are not limited to: Cas9, Csn2,and/or Cas4. In some embodiments, a Cas protein comprises Cas9, Csn2,and/or Cas4. In some embodiments, Examples of Cas proteins include, butare not limited to: Cas10, Csm2, Cmr5, Cas10, Csx11, and/or Csx10. Insome embodiments, a Cas protein comprises a Cas10, Csm2, Cmr5, Cas10,Csx11, and/or Csx10. In some embodiments, examples of Cas proteinsinclude, but are not limited to: Csf1. In some embodiments, a Casprotein comprises Csf1. In some embodiments, examples of Cas proteinsinclude, but are not limited to: Cas12a, Cas12b, Cas12c, C2c4, C2c8,C2c5, C2c10, and C2c9; as well as CasX (Cas12e) and CasY (Cas12d). Alsosee, e.g., Koonin et al., Curr Opin Microbiol. 2017; 37:67-78:“Diversity, classification and evolution of CRISPR-Cas systems.” In someembodiments, a Cas protein comprises Cas12a, Cas12b, Cas12c, Cas12d,Cas12e, Cas12d, and/or Cas12e. In some embodiments, a Cas proteincomprises Cas13, Cas13a, C2c2, Cas13b, Cas13c, and/or Cas13d. In someembodiments, the CRISPR/Cas system comprises a Cas effector proteinselected from the group consisting of: a) Cas3, Cas8a, Cas5, Cas8b,Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; b) Cas9, Csn2,and Cas4; c) Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; d) Csf1; e)Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e),and CasY (Cas12d); and f) Cas13, Cas13a, C2c2, Cas13b, Cas13c, andCas13d.

In some embodiments, a Cas protein comprises any one of the Cas proteinsdescribed herein or a functional portion thereof. As used herein,“functional portion” refers to a portion of a peptide which retains itsability to complex with at least one ribonucleic acid (e.g., guide RNA(gRNA)) and cleave a target polynucleotide sequence. In someembodiments, the functional portion comprises a combination of operablylinked Cas9 protein functional domains selected from the groupconsisting of a DNA binding domain, at least one RNA binding domain, ahelicase domain, and an endonuclease domain. In some embodiments, thefunctional portion comprises a combination of operably linked Cas12a(also known as Cpf1) protein functional domains selected from the groupconsisting of a DNA binding domain, at least one RNA binding domain, ahelicase domain, and an endonuclease domain. In some embodiments, thefunctional domains form a complex. In some embodiments, a functionalportion of the Cas9 protein comprises a functional portion of aRuvC-like domain. In some embodiments, a functional portion of the Cas9protein comprises a functional portion of the HNH nuclease domain. Insome embodiments, a functional portion of the Cas12a protein comprises afunctional portion of a RuvC-like domain.

In some embodiments, exogenous Cas protein can be introduced into thecell in polypeptide form. In certain embodiments, Cas proteins can beconjugated to or fused to a cell-penetrating polypeptide orcell-penetrating peptide. As used herein, “cell-penetrating polypeptide”and “cell-penetrating peptide” refers to a polypeptide or peptide,respectively, which facilitates the uptake of molecule into a cell. Thecell-penetrating polypeptides can contain a detectable label.

In many embodiments, Cas proteins can be conjugated to or fused to acharged protein (e.g., that carries a positive, negative or overallneutral electric charge). Such linkage may be covalent. In someembodiments, the Cas protein can be fused to a superpositively chargedGFP to significantly increase the ability of the Cas protein topenetrate a cell (Cronican et al. ACS Chem Biol. 2010; 5(8):747-52). Incertain embodiments, the Cas protein can be fused to a proteintransduction domain (PTD) to facilitate its entry into a cell. ExemplaryPTDs include Tat, oligoarginine, and penetratin. In some embodiments,the Cas9 protein comprises a Cas9 polypeptide fused to acell-penetrating peptide. In some embodiments, the Cas9 proteincomprises a Cas9 polypeptide fused to a PTD. In some embodiments, theCas9 protein comprises a Cas9 polypeptide fused to a tat domain. In someembodiments, the Cas9 protein comprises a Cas9 polypeptide fused to anoligoarginine domain. In some embodiments, the Cas9 protein comprises aCas9 polypeptide fused to a penetratin domain. In some embodiments, theCas9 protein comprises a Cas9 polypeptide fused to a superpositivelycharged GFP. In some embodiments, the Cas12a protein comprises a Cas12apolypeptide fused to a cell-penetrating peptide. In some embodiments,the Cas12a protein comprises a Cas12a polypeptide fused to a PTD. Insome embodiments, the Cas12a protein comprises a Cas12a polypeptidefused to a tat domain. In some embodiments, the Cas12a protein comprisesa Cas12a polypeptide fused to an oligoarginine domain. In someembodiments, the Cas12a protein comprises a Cas12a polypeptide fused toa penetratin domain. In some embodiments, the Cas12a protein comprises aCas12a polypeptide fused to a superpositively charged GFP.

In some embodiments, the Cas protein can be introduced into a cellcontaining the target polynucleotide sequence in the form of a nucleicacid encoding the Cas protein. The process of introducing the nucleicacids into cells can be achieved by any suitable technique. Suitabletechniques include calcium phosphate or lipid-mediated transfection,electroporation, and transduction or infection using a viral vector. Insome embodiments, the nucleic acid comprises DNA. In some embodiments,the nucleic acid comprises a modified DNA, as described herein. In someembodiments, the nucleic acid comprises mRNA. In some embodiments, thenucleic acid comprises a modified mRNA, as described herein (e.g., asynthetic, modified mRNA).

In some embodiments, the Cas protein is complexed with one to tworibonucleic acids. In some embodiments, the Cas protein is complexedwith two ribonucleic acids. In some embodiments, the Cas protein iscomplexed with one ribonucleic acid. In some embodiments, the Casprotein is encoded by a modified nucleic acid, as described herein(e.g., a synthetic, modified mRNA).

The methods of the present disclosure contemplate the use of anyribonucleic acid that is capable of directing a Cas protein to andhybridizing to a target motif of a target polynucleotide sequence. Insome embodiments, at least one of the ribonucleic acids comprisestracrRNA. In some embodiments, at least one of the ribonucleic acidscomprises CRISPR RNA (crRNA). In some embodiments, a single ribonucleicacid comprises a guide RNA that directs the Cas protein to andhybridizes to a target motif of the target polynucleotide sequence in acell. In some embodiments, at least one of the ribonucleic acidscomprises a guide RNA that directs the Cas protein to and hybridizes toa target motif of the target polynucleotide sequence in a cell. In someembodiments, both of the one to two ribonucleic acids comprise a guideRNA that directs the Cas protein to and hybridizes to a target motif ofthe target polynucleotide sequence in a cell. The ribonucleic acids ofthe present disclosure can be selected to hybridize to a variety ofdifferent target motifs, depending on the particular CRISPR/Cas systememployed, and the sequence of the target polynucleotide, as will beappreciated by those skilled in the art. The one to two ribonucleicacids can also be selected to minimize hybridization with nucleic acidsequences other than the target polynucleotide sequence. In someembodiments, the one to two ribonucleic acids hybridize to a targetmotif that contains at least two mismatches when compared with all othergenomic nucleotide sequences in the cell. In some embodiments, the oneto two ribonucleic acids hybridize to a target motif that contains atleast one mismatch when compared with all other genomic nucleotidesequences in the cell. In some embodiments, the one to two ribonucleicacids are designed to hybridize to a target motif immediately adjacentto a deoxyribonucleic acid motif recognized by the Cas protein. In someembodiments, each of the one to two ribonucleic acids are designed tohybridize to target motifs immediately adjacent to deoxyribonucleic acidmotifs recognized by the Cas protein which flank a mutant allele locatedbetween the target motifs.

In some embodiments, each of the one to two ribonucleic acids comprisesguide RNAs that directs the Cas protein to and hybridizes to a targetmotif of the target polynucleotide sequence in a cell.

In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) arecomplementary to and/or hybridize to sequences on the same strand of atarget polynucleotide sequence. In some embodiments, one or tworibonucleic acids (e.g., guide RNAs) are complementary to and/orhybridize to sequences on the opposite strands of a targetpolynucleotide sequence. In some embodiments, the one or two ribonucleicacids (e.g., guide RNAs) are not complementary to and/or do nothybridize to sequences on the opposite strands of a targetpolynucleotide sequence. In some embodiments, the one or two ribonucleicacids (e.g., guide RNAs) are complementary to and/or hybridize tooverlapping target motifs of a target polynucleotide sequence. In someembodiments, the one or two ribonucleic acids (e.g., guide RNAs) arecomplementary to and/or hybridize to offset target motifs of a targetpolynucleotide sequence.

In some embodiments, nucleic acids encoding Cas protein and nucleicacids encoding the at least one to two ribonucleic acids are introducedinto a cell via viral transduction (e.g., lentiviral transduction). Insome embodiments, the Cas protein is complexed with 1-2 ribonucleicacids. In some embodiments, the Cas protein is complexed with tworibonucleic acids. In some embodiments, the Cas protein is complexedwith one ribonucleic acid. In some embodiments, the Cas protein isencoded by a modified nucleic acid, as described herein (e.g., asynthetic, modified mRNA).

Exemplary gRNA sequences useful for CRISPR/Cas-based targeting of genesdescribed herein are provided in Table 15. The sequences can be found inWO2016183041 filed May 9, 2016, the disclosure including the Tables,Appendices, and Sequence Listing is incorporated herein by reference inits entirety.

TABLE 15 Exemplary gRNA sequences useful for targeting genes Gene NameSEQ ID NO: WO2016183041 HLA-A SEQ ID NOs: 2-1418 Table 8, Appendix 1HLA-B SEQ ID NOs: 1419-3277 Table 9, Appendix 2 HLA-C SEQ ID NOS:3278-5183 Table 10, Appendix 3 RFX-ANK SEQ ID NOs: 95636-102318 Table11, Appendix 4 NFY-A SEQ ID NOs: 102319-121796 Table 13, Appendix 6 RFX5SEQ ID NOs: 85645-90115 Table 16, Appendix 9 RFX-AP SEQ ID NOs:90116-95635 Table 17, Appendix 10 NFY-B SEQ ID NOs: 121797-135112 Table20, Appendix 13 NFY-C SEQ ID NOs: 135113-176601 Table 22, Appendix 15IRF1 SEQ ID NOs: 176602-182813 Table 23, Appendix 16 TAP1 SEQ ID NOs:182814-188371 Table 24, Appendix 17 CIITA SEQ ID NOS: 5184-36352 Table12, Appendix 5 B2M SEQ ID NOS: 81240-85644 Table 15, Appendix 8 NLRC5SEQ ID NOS: 36353-81239 Table 14, Appendix 7 CD47 SEQ ID NOS:200784-231885 Table 29, Appendix 22 HLA-E SEQ ID NOS: 189859-193183Table 19, Appendix 12 HLA-F SEQ ID NOS: 688808-699754 Table 45, Appendix38 HLA-G SEQ ID NOS: 188372-189858 Table 18, Appendix 11 PD-L1 SEQ IDNOS: 193184-200783 Table 21, Appendix 14 Gene Name SEQ ID NO:US20160348073 TRAC SEQ ID NOS: 532-609 and 9102-9797 Gene Name SEQ IDNO: WO2016183041 TRB (also SEQ ID NOS: 610-765 and 9798- TCRB and 10532TRBC)

Other exemplary gRNA sequences useful for CRISPR/Cas-based targeting ofgenes described herein are provided in U.S. Provisional PatentApplication No. 63/190,685, filed May 19, 2021, and in U.S. ProvisionalPatent Application No. 63/221,887, filed Jul. 14, 2021, the disclosuresof which, including the Tables, Appendices, and Sequence Listings, areincorporated herein by reference in their entireties.

In some embodiments, the cells of the technology are made usingTranscription Activator-Like Effector Nucleases (TALEN) methodologies.

By a “TALE-nuclease” (TALEN) is intended a fusion protein consisting ofa nucleic acid-binding domain typically derived from a TranscriptionActivator Like Effector (TALE) and one nuclease catalytic domain tocleave a nucleic acid target sequence. The catalytic domain ispreferably a nuclease domain and more preferably a domain havingendonuclease activity, like for instance I-TevI, ColE7, NucA and Fok-I.In numerous embodiments, the TALE domain can be fused to a meganucleaselike for instance I-CreI and I-OnuI or functional variant thereof. In amore preferred embodiment, said nuclease is a monomeric TALE-Nuclease. Amonomeric TALE-Nuclease is a TALE-Nuclease that does not requiredimerization for specific recognition and cleavage, such as the fusionsof engineered TAL repeats with the catalytic domain of I-TevI describedin WO2012138927. Transcription Activator like Effector (TALE) areproteins from the bacterial species Xanthomonas comprise a plurality ofrepeated sequences, each repeat comprising di-residues in position 12and 13 (RVD) that are specific to each nucleotide base of the nucleicacid targeted sequence. Binding domains with similar modularbase-per-base nucleic acid binding properties (MBBBD) can also bederived from new modular proteins recently discovered by the applicantin a different bacterial species. The new modular proteins have theadvantage of displaying more sequence variability than TAL repeats.Preferably, RVDs associated with recognition of the differentnucleotides are HD for recognizing C, NG for recognizing T, NI forrecognizing A, NN for recognizing G or A, NS for recognizing A, C, G orT, HG for recognizing T, IG for recognizing T, NK for recognizing G, HAfor recognizing C, ND for recognizing C, HI for recognizing C, HN forrecognizing G, NA for recognizing G, SN for recognizing G or A and YGfor recognizing T, TL for recognizing A, VT for recognizing A or G andSW for recognizing A. In another embodiment, critical amino acids 12 and13 can be mutated towards other amino acid residues in order to modulatetheir specificity towards nucleotides A, T, C and G and in particular toenhance this specificity. TALEN kits are sold commercially.

In some embodiments, the cells are manipulated using zinc fingernuclease (ZFN). A “zinc finger binding protein” is a protein orpolypeptide that binds DNA, RNA and/or protein, preferably in asequence-specific manner, as a result of stabilization of proteinstructure through coordination of a zinc ion. The term zinc fingerbinding protein is often abbreviated as zinc finger protein or ZFP. Theindividual DNA binding domains are typically referred to as “fingers.” AZFP has least one finger, typically two fingers, three fingers, or sixfingers. Each finger binds from two to four base pairs of DNA, typicallythree or four base pairs of DNA. A ZFP binds to a nucleic acid sequencecalled a target site or target segment. Each finger typically comprisesan approximately 30 amino acid, zinc-chelating, DNA-binding subdomain.Studies have demonstrated that a single zinc finger of this classconsists of an alpha helix containing the two invariant histidineresidues co-ordinated with zinc along with the two cysteine residues ofa single beta turn (see, e.g., Berg & Shi, Science 271:1081-1085(1996)).

In some embodiments, the cells of the present disclosure are made usinga homing endonuclease. Such homing endonucleases are well-known to theart (Stoddard 2005). Homing endonucleases recognize a DNA targetsequence and generate a single- or double-strand break. Homingendonucleases are highly specific, recognizing DNA target sites rangingfrom 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40bp in length. The homing endonuclease according to the technology mayfor example correspond to a LAGLIDADG endonuclease, to a HNHendonuclease, or to a GIY-YIG endonuclease. Preferred homingendonuclease according to the present disclosure can be an I-CreIvariant.

In some embodiments, the cells of the technology are made using ameganuclease. Meganucleases are by definition sequence-specificendonucleases recognizing large sequences (Chevalier, B. S. and B. L.Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). They can cleaveunique sites in living cells, thereby enhancing gene targeting by1000-fold or more in the vicinity of the cleavage site (Puchta et al.,Nucleic Acids Res., 1993, 21, 5034-5040; Rouet et al., Mol. Cell. Biol.,1994, 14, 8096-8106; Choulika et al., Mol. Cell. Biol., 1995, 15,1968-1973; Puchta et al., Proc. Natl. Acad. Sci. USA, 1996, 93,5055-5060; Sargent et al., Mol. Cell. Biol., 1997, 17, 267-77; Donoho etal., Mol. Cell. Biol, 1998, 18, 4070-4078; Elliott et al., Mol. Cell.Biol., 1998, 18, 93-101; Cohen-Tannoudji et al., Mol. Cell. Biol., 1998,18, 1444-1448).

In some embodiments, the cells of the technology are made using RNAsilencing or RNA interference (RNAi) to knock down (e.g., decrease,eliminate, or inhibit) the expression of a polypeptide such as atolerogenic factor. Useful RNAi methods include those that utilizesynthetic RNAi molecules, short interfering RNAs (siRNAs),PIWI-interacting NRAs (piRNAs), short hairpin RNAs (shRNAs), microRNAs(miRNAs), and other transient knock down methods recognized by thoseskilled in the art. Reagents for RNAi including sequence specificshRNAs, siRNA, miRNAs and the like are commercially available. Forinstance, CIITA can be knocked down in a pluripotent stem cell byintroducing a CIITA siRNA or transducing a CIITA shRNA-expressing virusinto the cell. In some embodiments, RNA interference is employed toreduce or inhibit the expression of at least one selected from the groupconsisting of CIITA, B2M, NLRC5, TCR-alpha, and TCR-beta.

In some embodiments, the cells provided herein are genetically modifiedto reduce expression of one or more immune factors (including targetpolypeptides) to create immune-privileged or hypoimmunogenic cells. Incertain embodiments, the cells (e.g., stem cells, induced pluripotentstem cells, differentiated cells, hematopoietic stem cells, primary Tcells and CAR-T cells) disclosed herein comprise one or more geneticmodifications to reduce expression of one or more targetpolynucleotides. Non-limiting examples of such target polynucleotidesand polypeptides include CIITA, B2M, NLRC5, CTLA-4, PD-1, HLA-A, HLA-BM,HLA-C, RFX-ANK, NFY-A, RFX5, RFX-AP, NFY-B, NFY-C, IRF1, and TAP1.

In some embodiments, the genetic modification occurs using a CRISPR/Cassystem. By modulating (e.g., reducing or deleting) expression of one ora plurality of the target polynucleotides, such cells exhibit decreasedimmune activation when engrafted into a recipient subject. In someembodiments, the cell is considered hypoimmunogenic, e.g., in arecipient subject or patient upon administration.

I. Gene Editing Systems

In some embodiments, the methods for genetically modifying cells toknock out, knock down, or otherwise modify one or more genes compriseusing a site-directed nuclease, including, for example, zinc fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs), meganucleases, transposases, and clustered regularlyinterspaced short palindromic repeat (CRISPR)/Cas systems, as well asnickase systems, base editing systems, prime editing systems, and genewriting systems known in the art.

i. ZFNs

ZFNs are fusion proteins comprising an array of site-specific DNAbinding domains adapted from zinc finger-containing transcriptionfactors attached to the endonuclease domain of the bacterial FokIrestriction enzyme. A ZFN may have one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more) of the DNA binding domains or zinc finger domains.See, e.g., Carroll et al., Genetics Society of America (2011)188:773-782; Kim et al., Proc. Natl. Acad. Sci. USA (1996) 93:1156-1160.Each zinc finger domain is a small protein structural motif stabilizedby one or more zinc ions and usually recognizes a 3- to 4-bp DNAsequence. Tandem domains can thus potentially bind to an extendednucleotide sequence that is unique within a cell's genome.

Various zinc fingers of known specificity can be combined to producemulti-finger polypeptides which recognize about 6, 9, 12, 15, or 18-bpsequences. Various selection and modular assembly techniques areavailable to generate zinc fingers (and combinations thereof)recognizing specific sequences, including phage display, yeastone-hybrid systems, bacterial one-hybrid and two-hybrid systems, andmammalian cells. Zinc fingers can be engineered to bind a predeterminednucleic acid sequence. Criteria to engineer a zinc finger to bind to apredetermined nucleic acid sequence are known in the art. See, e.g.,Sera et al., Biochemistry (2002) 41:7074-7081; Liu et al.,Bioinformatics (2008) 24:1850-1857.

ZFNs containing FokI nuclease domains or other dimeric nuclease domainsfunction as a dimer. Thus, a pair of ZFNs are required to targetnon-palindromic DNA sites. The two individual ZFNs must bind oppositestrands of the DNA with their nucleases properly spaced apart. SeeBitinaite et al., Proc. Natl. Acad. Sci. USA (1998) 95:10570-10575. Tocleave a specific site in the genome, a pair of ZFNs are designed torecognize two sequences flanking the site, one on the forward strand andthe other on the reverse strand. Upon binding of the ZFNs on either sideof the site, the nuclease domains dimerize and cleave the DNA at thesite, generating a DSB with 5′ overhangs. HDR can then be utilized tointroduce a specific mutation, with the help of a repair templatecontaining the desired mutation flanked by homology arms. The repairtemplate is usually an exogenous double-stranded DNA vector introducedto the cell. See Miller et al., Nat. Biotechnol. (2011) 29:143-148;Hockemeyer et al., Nat. Biotechnol. (2011) 29:731-734.

ii. TALENs

TALENs are another example of an artificial nuclease which can be usedto edit a target gene. TALENs are derived from DNA binding domainstermed TALE repeats, which usually comprise tandem arrays with 10 to 30repeats that bind and recognize extended DNA sequences. Each repeat is33 to 35 amino acids in length, with two adjacent amino acids (termedthe repeat-variable di-residue, or RVD) conferring specificity for oneof the four DNA base pairs. Thus, there is a one-to-one correspondencebetween the repeats and the base pairs in the target DNA sequences.

TALENs are produced artificially by fusing one or more TALE DNA bindingdomains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a nucleasedomain, for example, a FokI endonuclease domain. See Zhang, NatureBiotech. (2011) 29:149-153. Several mutations to FokI have been made forits use in TALENs; these, for example, improve cleavage specificity oractivity. See Cermak et al., Nucl. Acids Res. (2011) 39:e82; Miller etal., Nature Biotech. (2011) 29:143-148; Hockemeyer et al., NatureBiotech. (2011) 29:731-734; Wood et al., Science (2011) 333:307; Doyonet al., Nature Methods (2010) 8:74-79; Szczepek et al., Nature Biotech(2007) 25:786-793; Guo et al., J. Mol. Biol. (2010) 200:96. The FokIdomain functions as a dimer, requiring two constructs with unique DNAbinding domains for sites in the target genome with proper orientationand spacing. Both the number of amino acid residues between the TALE DNAbinding domain and the FokI nuclease domain and the number of basesbetween the two individual TALEN binding sites appear to be importantparameters for achieving high levels of activity. Miller et al., NatureBiotech. (2011) 29:143-148.

By combining engineered TALE repeats with a nuclease domain, asite-specific nuclease can be produced specific to any desired DNAsequence. Similar to ZFNs, TALENs can be introduced into a cell togenerate DSBs at a desired target site in the genome, and so can be usedto knock out genes or knock in mutations in similar, HDR-mediatedpathways. See Boch, Nature Biotech. (2011) 29:135-136; Boch et al.,Science (2009) 326:1509-1512; Moscou et al., Science (2009) 326:3501.

iii. Meganucleases

Meganucleases are enzymes in the endonuclease family which arecharacterized by their capacity to recognize and cut large DNA sequences(from 14 to 40 base pairs). Meganucleases are grouped into familiesbased on their structural motifs which affect nuclease activity and/orDNA recognition. The most widespread and best known meganucleases arethe proteins in the LAGLIDADG family, which owe their name to aconserved amino acid sequence. See Chevalier et al., Nucleic Acids Res.(2001) 29(18): 3757-3774. On the other hand, the GIY-YIG family membershave a GIY-YIG module, which is 70-100 residues long and includes fouror five conserved sequence motifs with four invariant residues, two ofwhich are required for activity. See Van Roey et al., Nature Struct.Biol. (2002) 9:806-811. The His-Cys family meganucleases arecharacterized by a highly conserved series of histidines and cysteinesover a region encompassing several hundred amino acid residues. SeeChevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774. Members ofthe NHN family are defined by motifs containing two pairs of conservedhistidines surrounded by asparagine residues. See Chevalier et al.,Nucleic Acids Res. (2001) 29(18):3757-3774.

Because the chance of identifying a natural meganuclease for aparticular target DNA sequence is low due to the high specificityrequirement, various methods including mutagenesis and high throughputscreening methods have been used to create meganuclease variants thatrecognize unique sequences. Strategies for engineering a meganucleasewith altered DNA-binding specificity, e.g., to bind to a predeterminednucleic acid sequence are known in the art. See, e.g., Chevalier et al.,Mol. Cell. (2002) 10:895-905; Epinat et al., Nucleic Acids Res (2003)31:2952-2962; Silva et al., J Mol. Biol. (2006) 361:744-754; Seligman etal., Nucleic Acids Res (2002) 30:3870-3879; Sussman et al., J Mol Biol(2004) 342:31-41; Doyon et al., J Am Chem Soc (2006) 128:2477-2484; Chenet al., Protein Eng Des Sel (2009) 22:249-256; Arnould et al., J MolBiol. (2006) 355:443-458; Smith et al., Nucleic Acids Res. (2006)363(2):283-294.

Like ZFNs and TALENs, Meganucleases can create DSBs in the genomic DNA,which can create a frame-shift mutation if improperly repaired, e.g.,via NHEJ, leading to a decrease in the expression of a target gene in acell. Alternatively, foreign DNA can be introduced into the cell alongwith the meganuclease. Depending on the sequences of the foreign DNA andchromosomal sequence, this process can be used to modify the targetgene. See Silva et al., Current Gene Therapy (2011) 11:11-27.

iv. Transposases

Transposases are enzymes that bind to the end of a transposon andcatalyze its movement to another part of the genome by a cut and pastemechanism or a replicative transposition mechanism. By linkingtransposases to other systems such as the CRISPER/Cas system, new geneediting tools can be developed to enable site specific insertions ormanipulations of the genomic DNA. There are two known DNA integrationmethods using transposons which use a catalytically inactive Caseffector protein and Tn7-like transposons. The transposase-dependent DNAintegration does not provoke DSBs in the genome, which may guaranteesafer and more specific DNA integration.

v. CRISPR Cas systems

The CRISPR system was originally discovered in prokaryotic organisms(e.g., bacteria and archaea) as a system involved in defense againstinvading phages and plasmids that provides a form of acquired immunity.Now it has been adapted and used as a popular gene editing tool inresearch and clinical applications.

CRISPR/Cas systems generally comprise at least two components: one ormore guide RNAs (gRNAs) and a Cas protein. The Cas protein is a nucleasethat introduces a DSB into the target site. CRISPR-Cas systems fall intotwo major classes: class 1 systems use a complex of multiple Casproteins to degrade nucleic acids; class 2 systems use a single largeCas protein for the same purpose. Class 1 is divided into types I, III,and IV; class 2 is divided into types II, V, and VI. Different Casproteins adapted for gene editing applications include, but are notlimited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas12,Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e(CasX), Cas12f (C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13,Cas13a (C2c2), Cas13b, Cas13c, Cas13d, C2c4, C2c8, C2c9, Cmr5, Cse1,Cse2, Csf1, Csm2, Csn2, Csx10, Csx11, Csy1, Csy2, Csy3, and Mad7. Themost widely used Cas9 is described herein as illustrative. These Casproteins may be originated from different source species. For example,Cas9 can be derived from S. pyogenes or S. aureus.

In the original microbial genome, the type II CRISPR system incorporatessequences from invading DNA between CRISPR repeat sequences encoded asarrays within the host genome. Transcripts from the CRISPR repeat arraysare processed into CRISPR RNAs (crRNAs) each harboring a variablesequence transcribed from the invading DNA, known as the “protospacer”sequence, as well as part of the CRISPR repeat. Each crRNA hybridizeswith a second transactivating CRISPR RNA (tracrRNA), and these two RNAsform a complex with the Cas9 nuclease. The protospacer-encoded portionof the crRNA directs the Cas9 complex to cleave complementary target DNAsequences, provided that they are adjacent to short sequences known as“protospacer adjacent motifs” (PAMs).

Since its discovery, the CRISPR system has been adapted for inducingsequence specific DSBs and targeted genome editing in a wide range ofcells and organisms spanning from bacteria to eukaryotic cells includinghuman cells. In its use in gene editing applications, artificiallydesigned, synthetic gRNAs have replaced the original crRNA:tracrRNAcomplex. For example, the gRNAs can be single guide RNAs (sgRNAs)composed of a crRNA, a tetraloop, and a tracrRNA. The crRNA usuallycomprises a complementary region (also called a spacer, usually about 20nucleotides in length) that is user-designed to recognize a target DNAof interest. The tracrRNA sequence comprises a scaffold region for Casnuclease binding. The crRNA sequence and the tracrRNA sequence arelinked by the tetraloop and each have a short repeat sequence forhybridization with each other, thus generating a chimeric sgRNA. One canchange the genomic target of the Cas nuclease by simply changing thespacer or complementary region sequence present in the gRNA. Thecomplementary region will direct the Cas nuclease to the target DNA sitethrough standard RNA-DNA complementary base pairing rules.

In order for the Cas nuclease to function, there must be a PAMimmediately downstream of the target sequence in the genomic DNA.Recognition of the PAM by the Cas protein is thought to destabilize theadjacent genomic sequence, allowing interrogation of the sequence by thegRNA and resulting in gRNA-DNA pairing when a matching sequence ispresent. The specific sequence of PAM varies depending on the species ofthe Cas gene. For example, the most commonly used Cas9 nuclease derivedfrom S. pyogenes recognizes a PAM sequence of 5′-NGG-3′ or, at lessefficient rates, 5′-NAG-3′, where “N” can be any nucleotide. Other Casnuclease variants with alternative PAMs have also been characterized andsuccessfully used for genome editing, which are summarized in Table 16below.

TABLE 16 Exemplary Cas nuclease variants and their PAM sequences CRISPRPAM Sequence Nuclease Source Organism (5′→3′) SpCas9Streptococcus pyogenes NGG or NAG SaCas9 Staphylococcus aureus NGRRT orNGRRN NmeCas9 Neisseria meningitidis NNNNGATT CjCas9Campylobacter jejuni NNNNRYAC StCas9 Streptococcus thermophilus NNAGAAWTdCas9 Treponema denticola NAAAAC LbCas12a Lachnospiraceae bacteriumTTTV (Cpf1) AsCas12a Acidaminococcus sp. TTTV (Cpf1) AacCas12bAlicyclobacillus TTN acidiphilus BhCas12b Bacillus hisashii ATTN, TTTN,v4 or GTTN R = A or G; Y = C or T; W = A or T; V = A or C or G; N = anybase

In some embodiments, Cas nucleases may comprise one or more mutations toalter their activity, specificity, recognition, and/or othercharacteristics. For example, the Cas nuclease may have one or moremutations that alter its fidelity to mitigate off-target effects (e.g.,eSpCas9, SpCas9-HF1, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelityvariants of SpCas9). For another example, the Cas nuclease may have oneor more mutations that alter its PAM specificity. 6. Nickases

Nuclease domains of the Cas, in particular the Cas9, nuclease can bemutated independently to generate enzymes referred to as DNA “nickases”.Nickases are capable of introducing a single-strand cut with the samespecificity as a regular CRISPR/Cas nuclease system, including forexample CRISPR/Cas9. Nickases can be employed to generate double-strandbreaks which can find use in gene editing systems (Mali et al., NatBiotech, 31(9):833-838 (2013); Mali et al. Nature Methods, 10:957-963(2013); Mali et al., Science, 339(6121):823-826 (2013)). In someinstances, when two Cas nickases are used, long overhangs are producedon each of the cleaved ends instead of blunt ends which allows foradditional control over precise gene integration and insertion (Mali etal., Nat Biotech, 31(9):833-838 (2013); Mali et al. Nature Methods,10:957-963 (2013); Mali et al., Science, 339(6121):823-826 (2013)). Asboth nicking Cas enzymes must effectively nick their target DNA, pairednickases can have lower off-target effects compared to thedouble-strand-cleaving Cas-based systems (Ran et al., Cell,155(2):479-480(2013); Mali et al., Nat Biotech, 31(9):833-838 (2013);Mali et al. Nature Methods, 10:957-963 (2013); Mali et al., Science,339(6121):823-826 (2013)).

S. Methods of Recombinant Expression of Tolerogenic Factors and/orChimeric Antigen Receptors

For all of these technologies, well-known recombinant techniques areused, to generate recombinant nucleic acids as outlined herein. Incertain embodiments, the recombinant nucleic acids encoding atolerogenic factor or a chimeric antigen receptor may be operably linkedto one or more regulatory nucleotide sequences in an expressionconstruct. Regulatory nucleotide sequences will generally be appropriatefor the host cell and recipient subject to be treated. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, the one or moreregulatory nucleotide sequences may include, but are not limited to,promoter sequences, leader or signal sequences, ribosomal binding sites,transcriptional start and termination sequences, translational start andtermination sequences, and enhancer or activator sequences. Constitutiveor inducible promoters as known in the art are also contemplated. Thepromoters may be either naturally occurring promoters, hybrid promotersthat combine elements of more than one promoter, or synthetic promoters.An expression construct may be present in a cell on an episome, such asa plasmid, or the expression construct may be inserted in a chromosomesuch as in a gene locus. In some embodiment, the expression vectorincludes a selectable marker gene to allow the selection of transformedhost cells. Some embodiments, include an expression vector comprising anucleotide sequence encoding a variant polypeptide operably linked to atleast one regulatory sequence. Regulatory sequence for use hereininclude promoters, enhancers, and other expression control elements. Insome embodiments, an expression vector is designed for the choice of thehost cell to be transformed, the particular variant polypeptide desiredto be expressed, the vector's copy number, the ability to control thatcopy number, and/or the expression of any other protein encoded by thevector, such as antibiotic markers.

Examples of suitable mammalian promoters include, for example, promotersfrom the following genes: elongation factor 1 alpha (EF1α) promoter, CAGpromoter, ubiquitin/S27a promoter of the hamster (WO 97/15664), Simianvacuolating virus 40 (SV40) early promoter, adenovirus major latepromoter, mouse metallothionein-I promoter, the long terminal repeatregion of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter(MMTV), Moloney murine leukemia virus Long Terminal repeat region, andthe early promoter of human Cytomegalovirus (CMV). Examples of otherheterologous mammalian promoters are the actin, immunoglobulin or heatshock promoter(s). In additional embodiments, promoters for use inmammalian host cells can be obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40). In furtherembodiments, heterologous mammalian promoters are used. Examples includethe actin promoter, an immunoglobulin promoter, and heat-shockpromoters. The early and late promoters of SV40 are convenientlyobtained as an SV40 restriction fragment which also contains the SV40viral origin of replication (Fiers et al., Nature 273: 113-120 (1978)).The immediate early promoter of the human cytomegalovirus isconveniently obtained as a HindIII restriction enzyme fragment(Greenaway et al., Gene 18: 355-360 (1982)). The foregoing referencesare incorporated by reference in their entirety.

In some embodiments, the expression vector is a bicistronic ormulticistronic expression vector. Bicistronic or multicistronicexpression vectors may include (1) multiple promoters fused to each ofthe open reading frames; (2) insertion of splicing signals betweengenes; (3) fusion of genes whose expressions are driven by a singlepromoter; and (4) insertion of proteolytic cleavage sites between genes(self-cleavage peptide) or insertion of internal ribosomal entry sites(IRESs) between genes.

The process of introducing the polynucleotides described herein intocells can be achieved by any suitable technique. Suitable techniquesinclude calcium phosphate or lipid-mediated transfection,electroporation, fusogens, and transduction or infection using a viralvector. In some embodiments, the polynucleotides are introduced into acell via viral transduction (e.g., AAV transduction, lentiviraltransduction) or otherwise delivered on a viral vector (e.g.,fusogen-mediated delivery). In some embodiments, the polynucleotides areintroduced into a cell via a fusogen-mediated delivery or a transposasesystem selected from the group consisting of conditional or inducibletransposases, conditional or inducible PiggyBac transposons, conditionalor inducible Sleeping Beauty (SB11) transposons, conditional orinducible Mos1 transposons, and conditional or inducible Tol2transposons.

In some embodiments, the cells provided herein are genetically modifiedto include one or more exogenous polynucleotides inserted into one ormore genomic loci of the hypoimmunogenic cell. In some embodiments, theexogenous polynucleotide encodes a protein of interest, e.g., a chimericantigen receptor. Any suitable method can be used to insert theexogenous polynucleotide into the genomic locus of the hypoimmunogeniccell including the gene editing methods described herein (e.g., aCRISPR/Cas system). In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction,for example, with a vector. In some embodiments, the vector is apseudotyped, self-inactivating lentiviral vector that carries theexogenous polynucleotide. In some embodiments, the vector is aself-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the exogenouspolynucleotide. In some embodiments, the exogenous polynucleotide isinserted into at least one allele of the cell using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into atleast one allele of the cell using a lentivirus based viral vector.

Unlike certain methods of introducing the polynucleotides describedherein into cells which generally involve activating cells, such asactivating T cells (e.g., CD8⁺ T cells), suitable techniques can beutilized to introduce polynucleotides into non-activated T cells.Suitable techniques include, but are not limited to, activation of Tcells, such as CD8⁺ T cells, with one or more antibodies which bind toCD3, CD8, and/or CD28, or fragments or portions thereof (e.g., scFv andVHH) that may or may not be bound to beads. Surprisingly,fusogen-mediated introduction of polynucleotides into T cells isperformed in non-activated T cells (e.g., CD8⁺ T cells) that have notbeen previously contacted with one or more activating antibodies orfragments or portions thereof (e.g., CD3, CD8, and/or CD28). In someembodiments, fusogen-mediated introduction of polynucleotides into Tcells is performed in vivo (e.g., after the T cells have beenadministered to a subject). In other embodiments, fusogen-mediatedintroduction of polynucleotides into T cells is performed in vitro(e.g., before the T cells are been administered to a subject).

Provided herein are non-activated T cells comprising reduced expressionof HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type T cell, wherein thenon-activated T cell further comprises a first exogenous polynucleotideencoding a chimeric antigen receptor (CAR).

In some embodiments, the non-activated T cell has not been treated withan anti-CD3 antibody, an anti-CD28 antibody, a T cell activatingcytokine, or a soluble T cell costimulatory molecule. In someembodiments, the non-activated T cell does not express activationmarkers. In some embodiments, the non-activated T cell expresses CD3 andCD28, and wherein the CD3 and/or CD28 are inactive.

In some embodiments, the anti-CD3 antibody is OKT3. In some embodiments,the anti-CD28 antibody is CD28.2. In some embodiments, the T cellactivating cytokine is selected from the group of T cell activatingcytokines consisting of IL-2, IL-7, IL-15, and IL-21. In someembodiments, the soluble T cell costimulatory molecule is selected fromthe group of soluble T cell costimulatory molecules consisting of ananti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, ananti-CD137L antibody, and an anti-ICOS-L antibody.

In some embodiments, the non-activated T cell is a primary T cell. Inother embodiments, the non-activated T cell is differentiated from thehypoimmunogenic cells of the present disclosure. In some embodiments,the T cell is a CD8⁺ T cell.

In some embodiments, the first exogenous polynucleotide encodesa CAR isselected from the group consisting of a CD19-specific CAR and aCD22-specific CAR.

In some embodiments, the first and/or second exogenous polynucleotide iscarried by a viral vector, including a lentiviral vector. In someembodiments, the first and/or second exogenous polynucleotide is carriedby a lentiviral vector that comprises a CD8 binding agent. In someembodiments, the first and/or second exogenous polynucleotide isintroduced into the cells using fusogen-mediated delivery or atransposase system selected from the group consisting of conditional orinducible transposases, conditional or inducible PiggyBac transposons,conditional or inducible Sleeping Beauty (SB11) transposons, conditionalor inducible Mos1 transposons, and conditional or inducible Tol2transposons.

In some embodiments, the non-activated T cell further comprises a secondexogenous polynucleotide encoding CD47. In some embodiments, the firstand/or second exogenous polynucleotides are inserted into a specificlocus of at least one allele of the T cell. In some embodiments, thespecific locus is selected from the group consisting of a safe harbor ortarget locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus,and a TRB locus. In some embodiments, the second exogenouspolynucleotide encoding CD47 is inserted into the specific locusselected from the group consisting of a safe harbor or target locus, atarget locus, a B2M locus, a CIITA locus, a TRAC locus and a TRB locus.In some embodiments, the first exogenous polynucleotide encoding the CARis inserted into the specific locus selected from the group consistingof a safe harbor or target locus, a target locus, a B2M locus, a CIITAlocus, a TRAC locus and a TRB locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into different loci. Insome embodiments, the second exogenous polynucleotide encoding CD47 andthe first exogenous polynucleotide encoding the CAR are inserted intothe same locus. In some embodiments, the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding the CARare inserted into the B2M locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into the CIITA locus. Insome embodiments, the second exogenous polynucleotide encoding CD47 andthe first exogenous polynucleotide encoding the CAR are inserted intothe TRAC locus. In some embodiments, the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding the CARare inserted into the TRB locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into the safe harbor ortarget locus. In some embodiments, the safe harbor or target locus isselected from the group consisting of a CCR5 gene locus, a CXCR4 genelocus, a PPP1R12C gene locus, an albumin gene locus, a SHS231 genelocus, a CLYBL gene locus, a Rosa gene locus, an F3 (CD142) gene locus,a MICA gene locus, a MICB gene locus, a LRP1 (CD91) gene locus, a HMGB1gene locus, an ABO gene locus, an RHD gene locus, a FUT1 locus, and aKDM5D gene locus.

In some embodiments, the non-activated T cell does not express HLA-A,HLA-B, and/or HLA-C antigens. In some embodiments, the non-activated Tcell does not express B2M. In some embodiments, the non-activated T celldoes not express HLA-DP, HLA-DQ, and/or HLA-DR antigens. In someembodiments, the non-activated T cell does not express CIITA. In someembodiments, the non-activated T cell does not express TCR-alpha. Insome embodiments, the non-activated T cell does not express TCR-beta. Insome embodiments, the non-activated T cell does not express TCR-alphaand TCR-beta.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the TRAC locus. In some embodiments, thenon-activated T cell is a B2M^(indel/indel), CIITA^(indel/indel),TRAC^(indel/indel) cell comprising the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding CARinserted into the TRAC locus. In some embodiments, the non-activated Tcell is a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel)cell comprising second exogenous polynucleotide encoding CD47 and/or thefirst exogenous polynucleotide encoding CAR inserted into the TRB locus.In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into the TRB locus. In someembodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the B2M locus. In some embodiments, thenon-activated T cell is a B2M^(indel/indel), CIITA^(indel/indel),TRAC^(indel/indel) cell comprising the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding CARinserted into a B2M locus. In some embodiments, the non-activated T cellis a B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising second exogenous polynucleotide encoding CD47 and/or thefirst exogenous polynucleotide encoding CAR inserted into the CIITAlocus. In some embodiments, the non-activated T cell is aB2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel) cellcomprising the second exogenous polynucleotide encoding CD47 and thefirst exogenous polynucleotide encoding CAR inserted into a CIITA locus.

In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRB^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the TRAC locus. In some embodiments, thenon-activated T cell is a B2M^(indel/indel), CIITA^(indel/indel),TRB^(indel/indel) cell comprising the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding CARinserted into the TRAC locus. In some embodiments, the non-activated Tcell is a B2M^(indel/indel), CIITA^(indel/indel), TRB^(indel/indel) cellcomprising second exogenous polynucleotide encoding CD47 and/or thefirst exogenous polynucleotide encoding CAR inserted into the TRB locus.In some embodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRB^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding CAR inserted into the TRB locus. In someembodiments, the non-activated T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRB^(indel/indel) cell comprising second exogenouspolynucleotide encoding CD47 and/or the first exogenous polynucleotideencoding CAR inserted into the B2M locus. In some embodiments, thenon-activated T cell is a B2M^(indel/indel), CIITA^(indel/indel),TRB^(indel/indel) cell comprising the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding CARinserted into a B2M locus. In some embodiments, the non-activated T cellis a B2M^(indel/indel), CIITA^(indel/indel), TRB^(indel/indel) cellcomprising second exogenous polynucleotide encoding CD47 and/or thefirst exogenous polynucleotide encoding CAR inserted into the CIITAlocus. In some embodiments, the non-activated T cell is aB2M^(indel/indel), CIITA^(indel/indel), TRB^(indel/indel) cellcomprising the second exogenous polynucleotide encoding CD47 and thefirst exogenous polynucleotide encoding CAR inserted into a CIITA locus.

Provided herein are engineered T cells comprising reduced expression ofHLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha,and/or TCR-beta relative to a wild-type T cell, wherein the engineered Tcell further comprises a first exogenous polynucleotide encoding achimeric antigen receptor (CAR) carried by a viral vector, including alentiviral vector. Provided herein are engineered T cells comprisingreduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M,CIITA, TCR-alpha, and/or TCR-beta relative to a wild-type T cell,wherein the engineered T cell further comprises a first exogenouspolynucleotide encoding a chimeric antigen receptor (CAR) carried by alentiviral vector that comprises a CD8 binding agent.

In some embodiments, the engineered T cell is a primary T cell. In otherembodiments, the engineered T cell is differentiated from thehypoimmunogenic cell of the present disclosure. In some embodiments, theT cell is a CD8⁺ T cell. In some embodiments, the T cell is a CD4⁺ Tcell.

In some embodiments, the engineered T cell does not express activationmarkers. In some embodiments, the engineered T cell expresses CD3 andCD28, and wherein the CD3 and/or CD28 are inactive.

In some embodiments, the engineered T cell has not been treated with ananti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine,or a soluble T cell costimulatory molecule. In some embodiments, theanti-CD3 antibody is OKT3, wherein the anti-CD28 antibody is CD28.2,wherein the T cell activating cytokine is selected from the group of Tcell activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21,and wherein soluble T cell costimulatory molecule is selected from thegroup of soluble T cell costimulatory molecules consisting of ananti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, ananti-CD137L antibody, and an anti-ICOS-L antibody. In some embodiments,the engineered T cell has not been treated with one or more T cellactivating cytokines selected from the group consisting of IL-2, IL-7,IL-15, and IL-21. In some instances, the cytokine is IL-2. In someembodiments, the one or more cytokines is IL-2 and another selected fromthe group consisting of IL-7, IL-15, and IL-21.

In some embodiments, the engineered T cell further comprises a secondexogenous polynucleotide encoding CD47. In some embodiments, the firstand/or second exogenous polynucleotides are inserted into a specificlocus of at least one allele of the T cell. In some embodiments, thespecific locus is selected from the group consisting of a safe harbor ortarget locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus,and a TRB locus. In some embodiments, the second exogenouspolynucleotide encoding CD47 is inserted into the specific locusselected from the group consisting of a safe harbor or target locus, atarget locus, a B2M locus, a CIITA locus, a TRAC locus and a TRB locus.In some embodiments, the first exogenous polynucleotide encoding the CARis inserted into the specific locus selected from the group consistingof a safe harbor or target locus, a target locus, a B2M locus, a CIITAlocus, a TRAC locus and a TRB locus. In some embodiments, the secondexogenous polynucleotide encoding CD47 and the first exogenouspolynucleotide encoding the CAR are inserted into different loci. Insome embodiments, the second exogenous polynucleotide encoding CD47 andthe first exogenous polynucleotide encoding the CAR are inserted intothe same locus. In some embodiments, the second exogenous polynucleotideencoding CD47 and the first exogenous polynucleotide encoding the CARare inserted into the B2M locus, the CIITA locus, the TRAC locus, theTRB locus, or the safe harbor or target locus. In some embodiments, thesafe harbor or target locus is selected from the group consisting of aCCR5 gene locus, a CXCR4 gene locus, a PPP1R12C gene locus, an albumingene locus, a SHS231 gene locus, a CLYBL gene locus, a Rosa gene locus,an F3 (CD142) gene locus, a MICA gene locus, a MICB gene locus, a LRP1(CD91) gene locus, a HMGB1 gene locus, an ABO gene locus, an RHD genelocus, a FUT1 locus, and a KDM5D gene locus.

In some embodiments, the CAR is selected from the group consisting of aCD19-specific CAR and a CD22-specific CAR. In some embodiments, the CARis a CD19-specific CAR. In some embodiments, the CAR is a CD22-specificCAR. In some embodiments, the CAR comprises an antigen binding domainthat binds to any one selected from the group consisting of CD19, CD22,CD38, CD123, CD138, and BCMA.

In some embodiments, the engineered T cell does not express HLA-A,HLA-B, and/or HLA-C antigens, wherein the engineered T cell does notexpress B2M, wherein the engineered T cell does not express HLA-DP,HLA-DQ, and/or HLA-DR antigens, wherein the engineered T cell does notexpress CIITA, and/or wherein the engineered T cell does not expressTCR-alpha and TCR-beta.

In some embodiments, the engineered T cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel) cell comprising the secondexogenous polynucleotide encoding CD47 and/or the first exogenouspolynucleotide encoding CAR inserted into the TRAC locus, into the TRBlocus, into the B2M locus, or into the CIITA locus. In some embodiments,the engineered T cell is a B2M^(indel/indel), CIITA^(indel/indel),TRB^(indel/indel) cell comprising the second exogenous polynucleotideencoding CD47 and/or the first exogenous polynucleotide encoding CARinserted into the TRAC locus, into the TRB locus, into the B2M locus, orinto the CIITA locus.

In some embodiments, the non-activated T cell and/or the engineered Tcell of the present disclosure are in a subject. In other embodiments,the non-activated T cell and/or the engineered T cell of the presentdisclosure are in vitro.

In some embodiments, the non-activated T cell and/or the engineered Tcell of the present disclosure express a CD8 binding agent. In someembodiments, the CD8 binding agent is an anti-CD8 antibody. In someembodiments, the anti-CD8 antibody is selected from the group consistingof a mouse anti-CD8 antibody, a rabbit anti-CD8 antibody, a humananti-CD8 antibody, a humanized anti-CD8 antibody, a camelid (e.g.,llama, alpaca, camel) anti-CD8 antibody, and a fragment thereof. In someembodiments, the fragment thereof is an scFv or a VHH. In someembodiments, the CD8 binding agent binds to a CD8 alpha chain and/or aCD8 beta chain.

In some embodiments, the CD8 binding agent is fused to a transmembranedomain incorporated in the viral envelope. In some embodiments, thelentivirus vector is pseudotyped with a viral fusion protein. In someembodiments, the viral fusion protein comprises one or moremodifications to reduce binding to its native receptor.

In some embodiments, the viral fusion protein is fused to the CD8binding agent. In some embodiments, the viral fusion protein comprisesNipah virus F glycoprotein and Nipah virus G glycoprotein fused to theCD8 binding agent. In some embodiments, the lentivirus vector does notcomprise a T cell activating molecule or a T cell costimulatorymolecule. In some embodiments, the lentivirus vector encodes the firstexogenous polynucleotide and/or the second exogenous polynucleotide.

In some embodiments, following transfer into a first subject, thenon-activated T cell or the engineered T cell exhibits one or moreresponses selected from the group consisting of (a) a T cell response,(b) an NK cell response, and (c) a macrophage response, that are reducedas compared to a wild-type cell following transfer into a secondsubject. In some embodiments, the first subject and the second subjectare different subjects. In some embodiments, the macrophage response isengulfment.

In some embodiments, following transfer into a subject, thenon-activated T cell or the engineered T cell exhibits one or moreselected from the group consisting of (a) reduced TH1 activation in thesubject, (b) reduced NK cell killing in the subject, and (c) reducedkilling by whole PBMCs in the subject, as compared to a wild-type cellfollowing transfer into the subject.

In some embodiments, following transfer into a subject, thenon-activated T cell or the engineered T cell elicits one or moreselected from the group consisting of (a) reduced donor specificantibodies in the subject, (b) reduced IgM or IgG antibodies in thesubject, and (c) reduced complement-dependent cytotoxicity (CDC) in asubject, as compared to a wild-type cell following transfer into thesubject.

In some embodiments, the non-activated T cell or the engineered T cellis transduced with a lentivirus vector comprising a CD8 binding agentwithin the subject. In some embodiments, the lentivirus vector carries agene encoding the CAR and/or CD47.

In some embodiments, the gene encoding the CAR and/or CD47 is introducedinto the cells using fusogen-mediated delivery, a transposase systemselected from the group consisting of transposases, PiggyBactransposons, Sleeping Beauty (SB11) transposons, Mos1 transposons, andTol2 transposons, or a viral vector, including a lentiviral vector.

Provided herein are pharmaceutical compositions comprising a populationof the non-activated T cells and/or the engineered T cells of thepresent disclosure and a pharmaceutically acceptable additive, carrier,diluent or excipient.

Provided herein are methods comprising administering to a subject acomposition comprising a population of the non-activated T cells and/orthe engineered T cells of the present disclosure, or one or more thepharmaceutical compositions of the present disclosure.

In some embodiments, the subject is not administered a T cell activatingtreatment before, after, and/or concurrently with administration of thecomposition. In some embodiments, the T cell activating treatmentcomprises lymphodepletion.

Provided herein are methods of treating a subject suffering from cancer,comprising administering to a subject a composition comprising apopulation of the non-activated T cells and/or the engineered T cells ofthe present disclosure, or one or more the pharmaceutical compositionsof the present disclosure, wherein the subject is not administered a Tcell activating treatment before, after, and/or concurrently withadministration of the composition. In some embodiments, the T cellactivating treatment comprises lymphodepletion.

Provided herein are methods for expanding T cells capable of recognizingand killing tumor cells in a subject in need thereof within the subject,comprising administering to a subject a composition comprising apopulation of the non-activated T cells and/or the engineered T cells ofthe present disclosure, or one or more the pharmaceutical compositionsof the present disclosure, wherein the subject is not administered a Tcell activating treatment before, after, and/or concurrently withadministration of the composition. In some embodiments, the T cellactivating treatment comprises lymphodepletion.

Provided herein are dosage regimens for treating a condition, disease ordisorder in a subject comprising administration of a pharmaceuticalcomposition comprising a population of the non-activated T cells and/orthe engineered T cells of the present disclosure, or one or more thepharmaceutical compositions of the present disclosure, and apharmaceutically acceptable additive, carrier, diluent or excipient,wherein the pharmaceutical composition is administered in about 1-3therapeutically effective doses. Provided herein are dosage regimens fortreating a condition, disease or disorder in a subject comprisingadministration of a pharmaceutical composition comprising a populationof the non-activated T cells and/or the engineered T cells of thepresent disclosure, or one or more the pharmaceutical compositions ofthe present disclosure, and a pharmaceutically acceptable additive,carrier, diluent or excipient, wherein the pharmaceutical composition isadministered in about 1-3 clinically effective doses.

Once altered, the presence of expression of any of the moleculedescribed herein can be assayed using known techniques, such as Westernblots, ELISA assays, FACS assays, other immunoassays, reversetranscriptase polymerase chain reactions (RT-PCR), and the like.

T. Generation of Induced Pluripotent Stem Cells

The technology provides methods of producing hypoimmunogenic pluripotentcells. In some embodiments, the method comprises generating pluripotentstem cells. The generation of mouse and human pluripotent stem cells(generally referred to as iPSCs; miPSCs for murine cells or hiPSCs forhuman cells) is generally known in the art. As will be appreciated bythose in the art, there are a variety of different methods for thegeneration of iPCSs. The original induction was done from mouseembryonic or adult fibroblasts using the viral introduction of fourtranscription factors, Oct3/4, Sox2, c-Myc and Klf4; see Takahashi andYamanaka Cell 126:663-676 (2006), hereby incorporated by reference inits entirety and specifically for the techniques outlined therein. Sincethen, a number of methods have been developed; see Seki et al, World J.Stem Cells 7(1): 116-125 (2015) for a review, and Lakshmipathy andVermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells,Methods and Protocols, Springer 2013, both of which are hereby expresslyincorporated by reference in their entirety, and in particular for themethods for generating hiPSCs (see for example Chapter 3 of the latterreference).

Generally, iPSCs are generated by the transient expression of one ormore reprogramming factors” in the host cell, usually introduced usingepisomal vectors. Under these conditions, small amounts of the cells areinduced to become iPSCs (in general, the efficiency of this step is low,as no selection markers are used). Once the cells are “reprogrammed”,and become pluripotent, they lose the episomal vector(s) and produce thefactors using the endogenous genes.

As is also appreciated by those of skill in the art, the number ofreprogramming factors that can be used or are used can vary. Commonly,when fewer reprogramming factors are used, the efficiency of thetransformation of the cells to a pluripotent state goes down, as well asthe “pluripotency”, e.g., fewer reprogramming factors may result incells that are not fully pluripotent but may only be able todifferentiate into fewer cell types.

In some embodiments, a single reprogramming factor, OCT4, is used. Inother embodiments, two reprogramming factors, OCT4 and KLF4, are used.In other embodiments, three reprogramming factors, OCT4, KLF4 and SOX2,are used. In other embodiments, four reprogramming factors, OCT4, KLF4,SOX2 and c-Myc, are used. In other embodiments, 5, 6 or 7 reprogrammingfactors can be used selected from SOKMNLT; SOX2, OCT4 (POU5F1), KLF4,MYC, NANOG, LIN28, and SV40L T antigen. In general, these reprogrammingfactor genes are provided on episomal vectors such as are known in theart and commercially available.

In general, as is known in the art, iPSCs are made from non-pluripotentcells such as, but not limited to, blood cells, fibroblasts, etc., bytransiently expressing the reprogramming factors as described herein.

U. Assays for Hypoimmunogenicity Phenotypes and Retention ofPluripotency

Once the hypoimmunogenic cells have been generated, they may be assayedfor their hypoimmunogenicity and/or retention of pluripotency as isdescribed in WO2016183041 and WO2018132783.

In some embodiments, hypoimmunogenicity is assayed using a number oftechniques as exemplified in FIG. 13 and FIG. 15 of WO2018132783. Thesetechniques include transplantation into allogeneic hosts and monitoringfor hypoimmunogenic pluripotent cell growth (e.g., teratomas) thatescape the host immune system. In some instances, hypoimmunogenicpluripotent cell derivatives are transduced to express luciferase andcan then followed using bioluminescence imaging. Similarly, the T celland/or B cell response of the host animal to such cells are tested toconfirm that the cells do not cause an immune reaction in the hostanimal. T cell responses can be assessed by Elispot, ELISA, FACS, PCR,or mass cytometry (CYTOF). B cell responses or antibody responses areassessed using FACS or Luminex. Additionally or alternatively, the cellsmay be assayed for their ability to avoid innate immune responses, e.g.,NK cell killing, as is generally shown in FIGS. 14 and 15 ofWO2018132783.

In some embodiments, the immunogenicity of the cells is evaluated usingT cell immunoassays such as T cell proliferation assays, T cellactivation assays, and T cell killing assays recognized by those skilledin the art. In some cases, the T cell proliferation assay includespretreating the cells with interferon-gamma and coculturing the cellswith labelled T cells and assaying the presence of the T cell population(or the proliferating T cell population) after a preselected amount oftime. In some cases, the T cell activation assay includes coculturing Tcells with the cells outlined herein and determining the expressionlevels of T cell activation markers in the T cells.

In vivo assays can be performed to assess the immunogenicity of thecells outlined herein. In some embodiments, the survival andimmunogenicity of hypoimmunogenic cells is determined using an allogenichumanized immunodeficient mouse model. In some instances, thehypoimmunogenic pluripotent stem cells are transplanted into anallogenic humanized NSG-SGM3 mouse and assayed for cell rejection, cellsurvival, and teratoma formation. In some instances, graftedhypoimmunogenic pluripotent stem cells or differentiated cells thereofdisplay long-term survival in the mouse model.

Additional techniques for determining immunogenicity includinghypoimmunogenicity of the cells are described in, for example, Deuse etal., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc NatlAcad Sci USA, 2019, 116(21), 10441-10446, the disclosures including thefigures, figure legends, and description of methods are incorporatedherein by reference in their entirety.

Similarly, the retention of pluripotency is tested in a number of ways.In some embodiments, pluripotency is assayed by the expression ofcertain pluripotency-specific factors as generally described herein andshown in FIG. 29 of WO2018132783. Additionally or alternatively, thepluripotent cells are differentiated into one or more cell types as anindication of pluripotency.

As will be appreciated by those in the art, the successful reduction ofthe MHC I function (HLA I when the cells are derived from human cells)in the pluripotent cells can be measured using techniques known in theart and as described below; for example, FACS techniques using labeledantibodies that bind the HLA complex; for example, using commerciallyavailable HLA-A, HLA-B, and HLA-C antibodies that bind to the alphachain of the human major histocompatibility HLA Class I antigens.

In addition, the cells can be tested to confirm that the HLA I complexis not expressed on the cell surface. This may be assayed by FACSanalysis using antibodies to one or more HLA cell surface components asdiscussed above.

The successful reduction of the MHC II function (HLA II when the cellsare derived from human cells) in the pluripotent cells or theirderivatives can be measured using techniques known in the art such asWestern blotting using antibodies to the protein, FACS techniques,RT-PCR techniques, etc.

In addition, the cells can be tested to confirm that the HLA II complexis not expressed on the cell surface. Again, this assay is done as isknown in the art (See FIG. 21 of WO2018132783, for example) andgenerally is done using either Western Blots or FACS analysis based oncommercial antibodies that bind to human HLA Class II HLA-DR, DP andmost DQ antigens.

In addition to the reduction of HLA I and II (or MHC I and II), thehypoimmunogenic cells of the technology have a reduced susceptibility tomacrophage phagocytosis and NK cell killing. The resultinghypoimmunogenic cells “escape” the immune macrophage and innate pathwaysdue to reduction or lack of the TCR complex and the expression of one ormore CD47 transgenes.

V. Exogenous Polynucleotides

In some embodiments, the hypoimmunogenic cells provided herein aregenetically modified to include one or more exogenous polynucleotidesinserted into one or more genomic loci of the hypoimmunogenic cell. Insome embodiments, the exogenous polynucleotide encodes a protein ofinterest, e.g., a chimeric antigen receptor. Any suitable method can beused to insert the exogenous polynucleotide into the genomic locus ofthe hypoimmunogenic cell including the gene editing methods describedherein (e.g., a CRISPR/Cas system). In some embodiments, the one or moreexogenous polynucleotides are inserted into at least one allele of thecell using viral transduction, for example, with a vector. In someembodiments, the vector is a pseudotyped, self-inactivating lentiviralvector that carries the one or more exogenous polynucleotides. In someembodiments, the vector is a self-inactivating lentiviral vectorpseudotyped with a vesicular stomatitis VSV-G envelope, and whichcarries the one or more exogenous polynucleotides. In some embodiments,the one or more exogenous polynucleotides are inserted into at least oneallele of the cell using viral transduction. In some embodiments, theone or more exogenous polynucleotide are inserted into at least oneallele of the cell using a lentivirus based viral vector.

The exogenous polynucleotide can be inserted into any suitable genomicloci of the hypoimmunogenic cell. In some embodiments, the exogenouspolynucleotide is inserted into a safe harbor or target locus asdescribed herein. Suitable safe harbor and target loci include, but arenot limited to, a CCR5 gene, a CXCR4 gene, a PPP1R12C (also known asAAVS1) gene, an albumin gene, a SHS231 locus, a CLYBL gene, a Rosa gene(e.g., ROSA26), an F3 gene (also known as CD142), a MICA gene, a MICBgene, a LRP1 gene (also known as CD91), a HMGB1 gene, an ABO gene, a RHDgene, a FUT1 gene, a PDGFRa gene, an OLIG2 gene, a GFAP gene, and aKDM5D gene (also known as HY). In some embodiments, the exogenouspolynucleotide is interested into an intron, exon, or coding sequenceregion of the safe harbor or target gene locus. In some embodiments, theexogenous polynucleotide is inserted into an endogenous gene wherein theinsertion causes silencing or reduced expression of the endogenous gene.In some embodiments, the polynucleotide is inserted in a B2M, CIITA,TRAC, TRB, PD-1 or CTLA-4 gene locus. Exemplary genomic loci forinsertion of an exogenous polynucleotide are depicted in Table 17.

TABLE 17 Exemplary genomic loci for insertion of exogenouspolynucleotides Target Also Num- region for known ber species NameEnsembl ID cleavage as 1 human B2M ENSG00000166710 CDS 2 human CIITAENSG00000179583 CDS 3 human TRAC ENSG00000277734 CDS 4 human PPP1R12CENSG00000125503 Intron 1 AAVS1 and 2 5 human CLYBL ENSG00000125246Intron 2 6 human CCR5 ENSG00000160791 Exons 1-3, introns 1-2, and CDS 7human THUMPD3- ENSG00000206573 Intron 1 ROSA26 AS1 8 human Ch- 500 bpSHS231 4:58,976,613 window 9 human F3 ENSG00000117525 CDS CD142 10 humanMICA ENSG00000204520 CDS 11 human MICB ENSG00000204516 CDS 12 human LRP1ENSG00000123384 CDS 13 human HMGB1 ENSG00000189403 CDS 14 human ABOENSG00000175164 CDS 15 human RHD ENSG00000187010 CDS 16 human FUT1ENSG00000174951 CDS 17 human KDM5D ENSG00000012817 CDS HY

TABLE 18 Non-limiting examples of Cas9 guide RNAs SEQ ID Target Gene NO:guide sequence PAM site gRNA cut location ABO 1 UCUCUCCAUGUGCAGUAGGA AGGExon chr9: 133,257,541 7 FUT1 2 CUGGAUGUCGGAGGAGUACG CGG Exonchr19: 48,750,822 4 RH 3 GUCUCCGGAAACUCGAGGUG AGG Exon chr1: 25,284,622F3 2 (CD142) 4 ACAGUGUAGACUUGAUUGAC GGG Exon chr1: 94,540,281 2 B2M 5CGUGAGUAAACCUGAAUCUU TGG Exon chr15: 44,715,434 2 CIITA 6GAUAUUGGCAUAAGCCUCCC TGG Exon chr16: 10,895,747 3 TRAC 7AGAGUCUCUCAGCUGGUACA CGG Exon chr14: 22,5547,533 1

For the Cas9 guides, the spacer sequence for all Cas9 guides is providedin Table 19, with description that the 20 nt guide sequence correspondsto a unique guide sequence and can be any of those described herein,including for example those listed in Table 18.

TABLE 19 Cas9 guide RNAs SEQ ID Description NO: Sequence 20 nt guide 8NNNNNNNNNNNNNNNNNNNN sequence* 12 nt crRNA 9 GUUUUAGAGCUA repeatsequence 4 nt 10 GAAA tetraloop sequence 64 nt 11UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU tracrRNAUGAAAAAGUGGCACCGAGUCGGUGCUUU sequence Exemplary 12NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAA fullAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC sequenceUUGAAAAAGUGGCACCGAGUCGGUGCUUU

In some embodiments, the hypoimmunogenic cell that includes theexogenous polynucleotide is derived from a hypoimmunogenic inducedpluripotent cell (HIP), for example, as described herein. Suchhypoimmunogenic cells include, for example, T cells and NIK cells. Insome embodiments, the hypoimmunogenic cell that includes the exogenouspolynucleotide is a T cell (e.g., a primary T cell), or an NK cell.

In some embodiments, the exogenous polynucleotide encodes an exogenousCD47 polypeptide (e.g., a human CD47 polypeptide) and the exogenouspolypeptide is inserted into the genome of the cell using a gene therapyvector. In some embodiments, the exogenous polynucleotide encodes anexogenous CD47 polypeptide (e.g., a human CD47 polypeptide) and theexogenous polypeptide is inserted into a safe harbor or target gene locior a safe harbor or target site as disclosed herein or a genomic locusthat causes silencing or reduced expression of the endogenous gene. Insome embodiments, the polynucleotide is inserted in a B2M, CIITA, TRAC,TRB, PD1 or CTLA4 gene locus.

In some embodiments, the hypoimmunogenic cell that includes theexogenous polynucleotide is a primary T cell or a T cell derived from ahypoimmunogenic pluripotent cell (e.g., a hypoimmunogenic iPSC). Inexemplary embodiments, the exogenous polynucleotide is a chimericantigen receptor (e.g., any of the CARs described herein). In someembodiments, the exogenous polynucleotide is operably linked to apromoter for expression of the exogenous polynucleotide in thehypoimmunogenic cell.

W. Pharmaceutically Acceptable Carriers

In some embodiments, the pharmaceutical composition provided hereinfurther include a pharmaceutically acceptable carrier. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); salts such assodium chloride; and/or non-ionic surfactants such as polysorbates(TWEEN™) poloxamers (PLURONICS™) or polyethylene glycol (PEG). In someembodiments, the pharmaceutical composition includes a pharmaceuticallyacceptable buffer (e.g., neutral buffer saline or phosphate bufferedsaline).

In some embodiments, the pharmaceutical composition includes one or moreelectrolyte base solutions selected from the group consisting oflactated CryoStor®, Ringer's solution, PlasmaLyte-A™, Iscove's ModifiedDulbecco's Medium, Normosol-R™, Veen-D™, Polysal® and Hank's BalancedSalt Solution (containing no phenol red). These base solutions closelyapproximate the composition of extracellular mammalian physiologicalfluids.

In some embodiments, the pharmaceutical composition includes one or morecryoprotective agents selected from the group consisting ofarabinogalactan, glycerol, polyvinylpyrrolidone (PVP), dextrose,dextran, trehalose, sucrose, raffinose, hydroxyethyl starch (HES),propylene glycol, human serum albumin (HSA), and dimethylsulfoxide(DMSO). In some embodiments, the pharmaceutically acceptable buffer isneutral buffer saline or phosphate buffered saline. In some embodiments,pharmaceutical compositions provided herein include one or more ofCryoStor® CSB, Plasma-Lyte-A™, HSA, DMSO, and trehalose.

CryoStor® is an intracellular-like optimized solution containingosmotic/oncotic agents, free radical scavengers, and energy sources tominimize apoptosis, minimize ischemia/reperfusion injury and maximizethe post-thaw recovery of the greatest numbers of viable, functionalcells. CryoStor® is serum- and protein-free, and non-immunogenic.CryoStor® is cGMP-manufactured from raw materials of USPgrade or higher.CryoStor® is a family of solutions pre-formulated with 0%, 2%, 5% or 10%DMSO. CryoStor® CSB is a DMSO-free version of CryoStor®. In someembodiments, the pharmaceutical composition includes a base solution ofCryoStor® CSB at a concentration of about 0-100%, 5-95%, 10-90%, 15-85%,20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 25-75%, 30-70%, 35-65%,40-60%, or 45-55% w/w. In some embodiments, the pharmaceuticalcomposition includes a base solution of CryoStor® CSB at a concentrationof about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% w/w.

PlasmaLyte-A™ is a non-polymeric plasma expander and contains essentialsalts and nutrients similar to those found in culture medium but doesnot contain additional constituents found in tissue culture medium whichare not approved for human infusion, e.g., phenol red, or areunavailable in U.S.P. grade. PlasmaLyte-A™ contains about 140 mEq/literof sodium (Na), about 5 mEq/liter of potassium (K), about 3 mEq/liter ofmagnesium (Mg), about 98 mEq/liter of chloride (Cl), about 27 mEq/literof acetate, and about 23 mEq/liter of gluconate. (PlasmaLyte-A™ iscommercially available from Baxter, Hyland Division, Glendale Calif.,product No. 2B2543). In some embodiments, the pharmaceutical compositionincludes a base solution of PlasmaLyte-A™ at a concentration of about0-100%, 5-95%, 10-90%, 15-85%, 15-80%, 15-75%, 15-70%, 15-65%, 15-60%,15-55%, 15-50%, 15-45%, 15-40%, 15-35%, 15-30%, 15-25%, 20-80%, 20-75%,20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%,25-75%, 30-70%, 35-65%, 40-60%, or 45-55% w/w. In some embodiments, thepharmaceutical composition includes a base solution of PlasmaLyte-A™ ata concentration of about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% w/w.

In some embodiments, the pharmaceutical composition includes human serumalbumin (HSA) at a concentration of about 0-10%, 0.3-9.3%, 0.3-8.3%,0.3-7.3%, 0.3-6.3%, 0.3-5.3%, 0.3-4.3%, 0.3-3.3%, 0.3-2.3%, 0.3-1.3%,0.6-8.3%, 0.9-7.3%, 1.2-6.3%, 1.5-5.3%, 1.8-4.3%, or 2.1-3.3% w/v. Insome embodiments, the pharmaceutical composition includes HSA at aconcentration of about 0%, 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, 1.8%, 2.1%,2.4%, 2.7%, 3.0%, 3.3%, 3.6%, 3.9%, 4.3%, 4.6%, 4.9%, 5.3%, 5.6%, 5.9%,6.3%, 6.6%, 6.9%, 7.3%, 7.6%, 7.9%, 8.3%, 8.6%, 8.9%, 9.3%, 9.6%, 9.9%,or 10% w/v.

In some embodiments, the pharmaceutical composition includesdimethylsulfoxide (DMSO) at a concentration of about 0-10%, 0.5-9.5%,1-9%, 1.5-8.5%, 2-8%, 3-8%, 4-8%, 5-8%, 6-8%, 7-8%, 2.5-7.5%, 3-7%,3.5-6.5%, 4-6%, or 4.5-5.5% v/v. In some embodiments, the pharmaceuticalcomposition includes HSA at a concentration of about 0%, 0.25%, 0.5%,0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%,3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%,6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%,9.0%, 9.25%, 9.5%, 9.75%, or 10.0% v/v.

In some embodiments, the pharmaceutical composition includes trehaloseat a concentration of about 0-500 mM, 50-450 mM, 100-400 mM, 150-350 mM,or 200-300 mM. In some embodiments, the pharmaceutical compositionincludes trehalose at a concentration of about 0 mM, 10 mM, 20 mM, 30mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM,175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM,400 mM, 425 mM, 450 mM, 475 mM, or 500 mM.

Exemplary pharmaceutical composition components are shown in Table 20.

TABLE 20 Exemplary pharmaceutical composition components. AdditionalFormulation Base Solution c[DMSO] c[HSA]* c[trehalose] A 75% CroStor ®7.5% 0.3% B CSB + 25% 3.75%  0.3% C PlasmaLyte A ™ + 5.3% D 1.2% HSA0.3% 250 mM E 100% 7.5% 0.3% F PlasmaLyte A ™ + 7.5% 5.3% G 1.2% HSA7.5% 5.3% 250 mM *Additional HSA in addition to PlasmaLyte.

In some embodiments, the pharmaceutical composition compriseshypoimmunogenic cells described herein and a pharmaceutically acceptablecarrier comprising 31.25% (v/v) Plasma-Lyte A, 31.25% (v/v) of 5%dextrose/0.45% sodium chloride, 10% dextran 40 (LMD)/5% dextrose, 20%(v/v) of 25% human serum albumin (HSA), and 7.5% (v/v) dimethylsulfoxide(DMSO).

X. Formulations and Dosage Regimens

Any therapeutically effective amount of cells described herein can beincluded in the pharmaceutical composition, depending on the indicationbeing treated. Non-limiting examples of the cells include primary Tcells, T cells differentiated from hypoimmunogenic induced pluripotentstem cells, and other cells differentiated from hypoimmunogenic inducedpluripotent stem cells described herein. In some embodiments, thepharmaceutical composition includes at least about 1×10², 5×10², 1×10³,5×10³, 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸,5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, or 5×10¹⁰ cells. In some embodiments, thepharmaceutical composition includes up to about 1×10², 5×10², 1×10³,5×10³, 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸,5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, or 5×10¹⁰ cells. In some embodiments, thepharmaceutical composition includes up to about 6.0×10⁸ cells. In someembodiments, the pharmaceutical composition includes up to about 8.0×10⁸cells. In some embodiments, the pharmaceutical composition includes atleast about 1×10²-5×10², 5×10²-1×10³, 1×10³-5×10³, 5×10³-1×10⁴,1×10⁴-5×10⁴, 5×10⁴-1×10⁵, 1×10⁵-5×10⁵, 5×10⁵-1×10⁶, 1×10⁶-5×10⁶,5×10⁶-1×10⁷, 1×10⁷-5×10⁷, 5×10⁷-1×10⁸, 1×10⁸-5×10⁸, 5×10⁹-1×10⁹,1×10⁹-5×10⁹, 5×10⁹-1×10¹⁰, or 1×10¹⁰-5×10¹⁰ cells. In exemplaryembodiments, the pharmaceutical composition includes from about 1.0×10⁶to about 2.5×10⁸ cells. In certain embodiments, the pharmaceuticalcomposition includes from about 2.0×10⁶ to about 2.0×10⁸ cells, such asbut not limited to, primary T cells, T cells differentiated fromhypoimmunogenic induced pluripotent stem cells.

In some embodiments, the pharmaceutical composition has a volume of atleast 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, or 500 ml. In exemplary embodiments, the pharmaceuticalcomposition has a volume of up to about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 ml. Inexemplary embodiments, the pharmaceutical composition has a volume ofabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, or 500 ml. In some embodiments, the pharmaceutical compositionhas a volume of from about 1-50 ml, 50-100 ml, 100-150 ml, 150-200 ml,200-250 ml, 250-300 ml, 300-350 ml, 350-400 ml, 400-450 ml, or 450-500ml. In some embodiments, the pharmaceutical composition has a volume offrom about 1-50 ml, 50-100 ml, 100-150 ml, 150-200 ml, 200-250 ml,250-300 ml, 300-350 ml, 350-400 ml, 400-450 ml, or 450-500 ml. In someembodiments, the pharmaceutical composition has a volume of from about1-10 ml, 10-20 ml, 20-30 ml, 30-40 ml, 40-50 ml, 50-60 ml, 60-70 ml,70-80 ml, 70-80 ml, 80-90 ml, or 90-100 ml. In some embodiments, thepharmaceutical composition has a volume that ranges from about 5 ml toabout 80 ml. In exemplary embodiments, the pharmaceutical compositionhas a volume that ranges from about 10 ml to about 70 ml. In certainembodiments, the pharmaceutical composition has a volume that rangesfrom about 10 ml to about 50 ml.

The specific amount/dosage regimen will vary depending on the weight,gender, age and health of the individual; the formulation, thebiochemical nature, bioactivity, bioavailability and the side effects ofthe cells and the number and identity of the cells in the completetherapeutic regimen.

In some embodiments, a therapeutically effective dose or a clinicallyeffective dose of the pharmaceutical composition includes about 1.0×10⁵to about 2.5×10⁸ cells at a volume of about 10 ml to 50 ml and thepharmaceutical composition is administered as a single therapeuticallyeffective dose or clinically effective dose. In some cases, thetherapeutically effective dose or clinically effective dose includesabout 1.0×10⁵ to about 2.5×10⁸ primary T cells described herein at avolume of about 10 ml to 50 ml. In some cases, the therapeuticallyeffective dose or clinically effective dose includes about 1.0×10⁵ toabout 2.5×10⁸ primary T cells that have been described above at a volumeof about 10 ml to 50 ml. In various cases, the therapeutically effectivedose or clinically effective dose includes about 1.0×10⁵ to about2.5×10⁸ T cells differentiated from hypoimmunogenic induced pluripotentstem cells described herein at a volume of about 10 ml to 50 ml. In someembodiments, the therapeutically effective dose or clinically effectivedose is 1.0×10⁵, 1.1×10⁵, 1.2×10⁵, 1.3×10⁵, 1.4×10⁵, 1.5×10⁵, 1.6×10⁵,1.7×10⁵, 1.8×10⁵, 1.9×10⁵, 2.0×10⁵, 2.1×10⁵, 2.2×10⁵, 2.3×10⁵, 2.4×10⁵,2.5×10⁵, 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶,1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶, 2.2×10⁶, 2.3×10⁶, 2.4×10⁶,2.5×10⁶, 1.0×10⁷, 1.1×10⁷, 1.2×10⁷, 1.3×10⁷, 1.4×10⁷, 1.5×10⁷, 1.6×10⁷,1.7×10⁷, 1.8×10⁷, 1.9×10⁷, 2.0×10⁷, 2.1×10⁷, 2.2×10⁷, 2.3×10⁷, 2.4×10⁷,2.5×10⁷, 1.0×10⁸, 1.1×10⁸, 1.2×10⁸, 1.3×10⁸, 1.4×10⁸, 1.5×10⁸, 1.6×10⁸,1.7×10⁸, 1.8×10⁸, 1.9×10⁸, 2.0×10⁸, 2.1×10⁸, 2.2×10⁸, 2.3×10⁸, 2.4×10⁸,or 2.5×10⁸ T cells differentiated from hypoimmunogenic inducedpluripotent stem cells described herein at a volume of about 10 ml to 50ml. In other cases, the therapeutically effective dose or clinicallyeffective dose is at a range that is lower than about 1.0×10⁵ to about2.5×10⁸ T cells, including primary T cells or T cells differentiatedfrom hypoimmunogenic induced pluripotent stem cells. In yet other cases,the therapeutically effective dose or clinically effective dose is at arange that is higher than about 1.0×10⁵ to about 2.5×10⁸ T cells,including primary T cells and T cells differentiated fromhypoimmunogenic induced pluripotent stem cells.

In some embodiments, the pharmaceutical composition is administered as asingle therapeutically effective dose or clinically effective dose offrom about 1.0×10⁵ to about 1.0×10⁷ cells (such as primary T cells and Tcells differentiated from hypoimmunogenic induced pluripotent stemcells) per kg body weight for subjects 50 kg or less. In someembodiments, the pharmaceutical composition is administered as a singletherapeutically effective dose or clinically effective dose of fromabout 0.5×10⁵ to about 1.0×10⁷, about 1.0×10⁵ to about 1.0×10⁷, about1.0×10⁵ to about 1.0×10⁷, about 5.0×10⁵ to about 1×10⁷, about 1.0×10⁶ toabout 1×10⁷, about 5.0×10⁶ to about 1.0×10⁷, about 1.0×10⁵ to about5.0×10⁶, about 1.0×10⁵ to about 1.0×10⁶, about 1.0×10⁵ to about 5.0×10⁵,about 1.0×10⁵ to about 5.0×10⁶, about 2.0×10⁵ to about 5.0×10⁶, about3.0×10⁵ to about 5.0×10⁶, about 4.0×10⁵ to about 5.0×10⁶, about 5.0×10⁵to about 5.0×10⁶, about 6.0×10⁵ to about 5.0×10⁶, about 7.0×10⁵ to about5.0×10⁶, about 8.0×10⁵ to about 5.0×10⁶, or about 9.0×10⁵ to about5.0×10⁶ cells per kg body weight for subjects 50 kg or less. In someembodiments, the therapeutically effective dose or clinically effectivedose is 0.5×10⁵, 0.6×10⁵, 0.7×10⁵, 0.8×10⁵, 0.9×10⁵, 1.0×10⁵, 1.1×10⁵,1.2×10⁵, 1.3×10⁵, 1.4×10⁵, 1.5×10⁵, 1.6×10⁵, 1.7×10⁵, 1.8×10⁵, 1.9×10⁵,2.0×10⁵, 2.1×10⁵, 2.2×10⁵, 2.3×10⁵, 2.4×10⁵, 2.5×10⁵, 2.6×10⁵, 2.7×10⁵,2.8×10⁵, 2.9×10⁵, 3.0×10⁵, 3.1×10⁵, 3.2×10⁵, 3.3×10⁵, 3.4×10⁵, 3.5×10⁵,3.6×10⁵, 3.7×10⁵, 3.8×10⁵, 3.9×10⁵, 4.0×10⁵, 4.1×10⁵, 4.2×10⁵, 4.3×10⁵,4.4×10⁵, 4.5×10⁵, 4.6×10⁵, 4.7×10⁵, 2.1×10⁵, 4.9×10⁵, 5.0×10⁵, 0.5×10⁶,0.6×10⁶, 0.7×10⁶, 0.8×10⁶, 0.9×10⁶, 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶,1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶,2.2×106, 2.3×10⁶, 2.4×10⁶, 2.5×10⁶, 2.6×10⁶, 2.7×10⁶, 2.8×10⁶, 2.9×10⁶,3.0×10⁶, 3.1×10⁶, 3.2×10⁶, 3.3×10⁶, 3.4×10⁶, 3.5×10⁶, 3.6×10⁶, 3.7×10⁶,3.8×10⁶, 3.9×10⁶, 4.0×10⁶, 4.1×106, 4.2×10⁶, 4.3×10⁶, 4.4×10⁶, 4.5×10⁶,4.6×10⁶, 4.7×10⁶, 4.8×10⁶, 4.9×10⁶, 5.0×10⁶, 5.1×10⁶, 5.2×10⁶, 5.3×10⁶,5.4×10⁶, 5.5×10⁶, 5.6×10⁶, 5.7×10⁶, 5.8×10⁶, 5.9×10⁶, 6.0×106, 6.1×10⁶,6.2×10⁶, 6.3×10⁶, 6.4×10⁶, 6.5×10⁶, 6.6×10⁶, 6.7×10⁶, 6.8×10⁶, 6.9×10⁶,7.0×10⁶, 7.1×10⁶, 7.2×10⁶, 7.3×10⁶, 7.4×10⁶, 7.5×10⁶, 7.6×10⁶, 7.7×10⁶,7.8×10⁶, 7.9×10⁶, 8.0×10⁶, 8.1×10⁶, 8.2×10⁶, 8.3×10⁶, 8.4×10⁶, 8.5×10⁶,8.6×10⁶, 8.7×10⁶, 8.8×10⁶, 8.9×10⁶, 9.0×10⁶, 9.1×10⁶, 9.2×10⁶, 9.3×10⁶,9.4×10⁶, 9.5×10⁶, 9.6×10⁶, 9.7×10⁶, 9.8×10⁶, 9.9×10⁶, 0.5×10⁷, 0.6×10⁷,0.7×10⁷, 0.8×10⁷, 0.9×10⁷, or 1.0×10⁷ cells per kg body weight forsubjects 50 kg or less. In some embodiments, the therapeuticallyeffective dose or clinically effective dose is from about 0.2×10⁶ toabout 5.0×10⁶ cells per kg body weight for subjects 50 kg or less. Incertain embodiments, the therapeutically effective dose or clinicallyeffective dose is at a range that is lower than from about 0.2×10⁶ toabout 5.0×10⁶ cells per kg body weight for subjects 50 kg or less. orclinically effective dose In exemplary embodiments, the singletherapeutically effective dose or clinically effective dose is at avolume of about 10 ml to 50 ml. In some embodiments, the therapeuticallyeffective dose or clinically effective dose is administeredintravenously.

In exemplary embodiments, the cells are administered in a singletherapeutically effective dose of from about 1.0×10⁶ to about 5.0×10⁸cells (such as primary T cells and T cells differentiated fromhypoimmunogenic induced pluripotent stem cells) for subjects above 50kg. In some embodiments, the pharmaceutical composition is administeredas a single therapeutically effective dose or clinically effective doseof from about 0.5×10⁶ to about 1.0×109, about 1.0×10⁶ to about 1.0×10⁹,about 1.0×10⁶ to about 1.0×10⁹, about 5.0×10⁶ to about 1.0×10⁹, about1.0×10⁷ to about 1.0×10⁹, about 5.0×10⁷ to about 1.0×10⁹, about 1.0×10⁶to about 5.0×10⁷, about 1.0×10⁶ to about 1.0×10⁷, about 1.0×10⁶ to about5.0×10⁷, about 1.0×10⁷ to about 5.0×10⁸, about 2.0×10⁷ to about 5.0×10⁸,about 3.0×10⁷ to about 5.0×10⁸, about 4.0×10⁷ to about 5.0×10⁸, about5.0×10⁷ to about 5.0×10⁸, about 6.0×10⁷ to about 5.0×10⁸, about 7.0×10⁷to about 5.0×10⁸, about 8.0×10⁷ to about 5.0×10⁸, or about 9.0×10⁷ toabout 5.0×10⁸ cells per kg body weight for subjects 50 kg or less. Insome embodiments, the therapeutically effective dose or clinicallyeffective dose is 1.0×10⁶, 1.1×106, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶,1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶, 2.2×10⁶, 2.3×10⁶,2.4×10⁶, 2.5×10⁶, 2.6×10⁶, 2.7×10⁶, 2.8×10⁶, 2.9×10⁶, 3.0×106, 3.1×10⁶,3.2×10⁶, 3.3×10⁶, 3.4×10⁶, 3.5×10⁶, 3.6×10⁶, 3.7×10⁶, 3.8×10⁶, 3.9×10⁶,4.0×10⁶, 4.1×10⁶, 4.2×10⁶, 4.3×10⁶, 4.4×10⁶, 4.5×10⁶, 4.6×10⁶, 4.7×10⁶,4.8×10⁶, 4.9×106, 5.0×10⁶, 5.1×10⁶, 5.2×10⁶, 5.3×10⁶, 5.4×10⁶, 5.5×10⁶,5.6×10⁶, 5.7×10⁶, 5.8×10⁶, 5.9×10⁶, 6.0×10⁶, 6.1×10⁶, 6.2×10⁶, 6.3×10⁶,6.4×10⁶, 6.5×10⁶, 6.6×10⁶, 6.7×10⁶, 6.8×106, 6.9×10⁶, 7.0×10⁶, 7.1×10⁶,7.2×10⁶, 7.3×10⁶, 7.4×10⁶, 7.5×10⁶, 7.6×10⁶, 7.7×10⁶, 7.8×10⁶, 7.9×10⁶,8.0×10⁶, 8.1×10⁶, 8.2×10⁶, 8.3×10⁶, 8.4×10⁶, 8.5×10⁶, 8.6×10⁶, 8.7×106,8.8×10⁶, 8.9×10⁶, 9.0×10⁶, 9.1×10⁶, 9.2×10⁶, 9.3×10⁶, 9.4×10⁶, 9.5×10⁶,9.6×10⁶, 9.7×10⁶, 9.8×10⁶, 9.9×10⁶, 1.0×10⁷, 1.1×10⁷, 1.2×10⁷, 1.3×10⁷,1.4×10⁷, 1.5×10⁷, 1.6×107, 1.7×10⁷, 1.8×10⁷, 1.9×10⁷, 2.0×10⁷, 2.1×10⁷,2.2×10⁷, 2.3×10⁷, 2.4×10⁷, 2.5×10⁷, 2.6×10⁷, 2.7×10⁷, 2.8×10⁷, 2.9×10⁷,3.0×10⁷, 3.1×10⁷, 3.2×10⁷, 3.3×10⁷, 3.4×10⁷, 3.5×107, 3.6×10⁷, 3.7×10⁷,3.8×10⁷, 3.9×10⁷, 4.0×10⁷, 4.1×10⁷, 4.2×10⁷, 4.3×10⁷, 4.4×10⁷, 4.5×10⁷,4.6×10⁷, 4.7×10⁷, 4.8×10⁷, 4.9×10⁷, 5.0×10⁷, 5.1×10⁷, 5.2×10⁷, 5.3×10⁷,5.4×107, 5.5×10⁷, 5.6×10⁷, 5.7×10⁷, 5.8×10⁷, 5.9×10⁷, 6.0×10⁷, 6.1×10⁷,6.2×10⁷, 6.3×10⁷, 6.4×10⁷, 6.5×10⁷, 6.6×10⁷, 6.7×10⁷, 6.8×10⁷, 6.9×10⁷,7.0×10⁷, 7.1×10⁷, 7.2×10⁷, 7.3×107, 7.4×10⁷, 7.5×10⁷, 7.6×10⁷, 7.7×10⁷,7.8×10⁷, 7.9×10⁷, 8.0×10⁷, 8.1×10⁷, 8.2×10⁷, 8.3×10⁷, 8.4×10⁷, 8.5×10⁷,8.6×10⁷, 8.7×10⁷, 8.8×10⁷, 8.9×10⁷, 9.0×10⁷, 9.1×10⁷, 9.2×107, 9.3×10⁷,9.4×10⁷, 9.5×10⁷, 9.6×10⁷, 9.7×10⁷, 9.8×10⁷, 9.9×10⁷, 1.0×10⁸, 1.1×10⁸,1.2×10⁸, 1.3×10⁸, 1.4×10⁸, 1.5×10⁸, 1.6×10⁸, 1.7×10⁸, 1.8×10⁸, 1.9×10⁸,2.0×10⁸, 2.1×10⁸, 2.2×10⁸, 2.3×10⁸, 2.4×10⁸, 2.5×10⁸, 2.6×10⁸, 2.7×10⁸,2.8×10⁸, 2.9×10⁸, 3.0×10⁸, 3.1×10⁸, 3.2×10⁸, 3.3×10⁸, 3.4×10⁸, 3.5×10⁸,3.6×10⁸, 3.7×10⁸, 3.8×10⁸, 3.9×10⁸, 4.0×10⁸, 4.1×10⁸, 4.2×10⁸, 4.3×10⁸,4.4×10⁸, 4.5×10⁸, 4.6×10⁸, 4.7×10⁸, 4.8×10⁸, 4.9×10⁸, or 5.0×10⁸ cellsper kg body weight for subjects 50 kg or less. In certain embodiments,the cells are administered in a single therapeutically effective dose orclinically effective dose of about 1.0×10⁷ to about 2.5×10⁸ cells forsubjects above 50 kg. In some embodiments, the cells are administered ina single therapeutically effective dose or clinically effective dose ofa range that is less than about 1.0×10⁷ to about 2.5×10⁸ cells forsubjects above 50 kg. In some embodiments, the cells are administered ina single therapeutically effective dose or clinically effective dose ofa range that is higher than about 1.0×10⁷ to about 2.5×10⁸ cells forsubjects above 50 kg. In some embodiments, the dose is administeredintravenously. In exemplary embodiments, the single therapeuticallyeffective dose or clinically effective dose is at a volume of about 10ml to 50 ml. In some embodiments, the therapeutically effective dose orclinically effective dose is administered intravenously.

In exemplary embodiments, the therapeutically effective dose orclinically effective dose is administered intravenously at a rate ofabout 1 to 50 ml per minute, 1 to 40 ml per minute, 1 to 30 ml perminute, 1 to 20 ml per minute, 10 to 20 ml per minute, 10 to 30 ml perminute, 10 to 40 ml per minute, 10 to 50 ml per minute, 20 to 50 ml perminute, 30 to 50 ml per minute, 40 to 50 ml per minute. In numerousembodiments, the pharmaceutical composition is stored in one or moreinfusion bags for intravenous administration. In some embodiments, thedose is administered completely at no more than 10 minutes, 15 minutes,20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes,50 minutes, 55 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes,120 minutes, 150 minutes, 180 minutes, 240 minutes, or 300 minutes.

In some embodiments, a single therapeutically effective dose orclinically effective dose of the pharmaceutical composition is presentin a single infusion bag. In other embodiments, a single therapeuticallyeffective dose or clinically effective dose of the pharmaceuticalcomposition is divided into 2, 3, 4 or 5 separate infusion bags.

In some embodiments, the cells described herein are administered in aplurality of doses such as 2, 3, 4, 5, 6 or more doses, wherein theplurality of doses together constitute a therapeutically effective doseor clinically effective dose regimen. In some embodiments, each dose ofthe plurality of doses is administered to the subject ranging from 1 to24 hours apart. In some instances, a subsequent dose is administeredfrom about 1 hour to about 24 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or about 24hours) after an initial or preceding dose. In some embodiments, eachdose of the plurality of doses is administered to the subject rangingfrom about 1 day to 28 days apart. In some instances, a subsequent doseis administered from about 1 day to about 28 days (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, or about 28 days) after an initial or precedingdose. In certain embodiments, each dose of the plurality of doses isadministered to the subject ranging from 1 week to about 6 weeks apart.In certain instances, a subsequent dose is administered from about 1week to about 6 weeks (e.g., about 1, 2, 3, 4, 5, or 6 weeks) after aninitial or preceding dose. In several embodiments, each dose of theplurality of doses is administered to the subject ranging from about 1month to about 12 months apart. In several instances, a subsequent doseis administered from about 1 month to about 12 months (e.g., about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) after an initial or precedingdose.

In some embodiments, a subject is administered a first dosage regimen ata first timepoint, and then subsequently administered a second dosageregimen at a second timepoint. In some embodiments, the first dosageregimen is the same as the second dosage regimen. In other embodiments,the first dosage regimen is different than the second dosage regimen. Insome instances, the number of cells in the first dosage regimen and thesecond dosage regimen are the same. In some instances, the number ofcells in the first dosage regimen and the second dosage regimen aredifferent. In some cases, the number of doses of the first dosageregimen and the second dosage regimen are the same. In some cases, thenumber of doses of the first dosage regimen and the second dosageregimen are different.

In some embodiments, the first dosage regimen includes hypoimmune (HIP)T cells or primary T cells expressing a first CAR and the second dosageregimen includes hypoimmune (HIP) T cells or primary T cells expressinga second CAR such that the first CAR and the second CAR are different.For instance, the first CAR and second CAR bind different targetantigens. In some cases, the first CAR includes an scFv that binds anantigen and the second CAR includes an scFv that binds a differentantigen. In some embodiments, the first dosage regimen includeshypoimmune (HIP) T cell or primary T cells expressing a first CAR andthe second dosage regimen includes hypoimmune (HIP) T cell or primary Tcells expressing a second CAR such that the first CAR and the second CARare the same. The first dosage regimen can be administered to thesubject at least 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months,1-3 months, 1-6 months, 4-6 months, 3-9 months, 3-12 months, or moremonths apart from the second dosage regimen. In some embodiments, asubject is administered a plurality of dosage regimens during the courseof a disease (e.g., cancer) and at least two of the dosage regimenscomprise the same type of hypoimmune (HIP) T cells or primary T cellsdescribed herein. In other embodiments, at least two of the plurality ofdosage regimens comprise different types of hypoimmune (HIP) T cells orprimary T cells described herein.

In some embodiments, the CD19 specific (CD19) CAR-T cells describedherein are administered to a subject at a dose of about 50×10⁶ to about110×10⁶ (e.g., 50×10⁶, 51×10⁶, 52×10⁶, 53×10⁶, 54×10⁶, 55×10⁶, 56×10⁶,57×10⁶, 58×10⁶, 59×10⁶, 60×10⁶, 61×10⁶, 62×10⁶, 63×10⁶, 64×10⁶, 65×10⁶,66×10⁶, 67×10⁶, 68×10⁶, 69×10⁶, 70×10⁶, 71×10⁶, 72×10⁶, 73×10⁶, 74×10⁶,75×10⁶, 76×10⁶, 77×10⁶, 78×10⁶, 79×10⁶, 80×10⁶, 81×10⁶, 82×10⁶, 83×10⁶,84×10⁶, 85×10⁶, 86×10⁶, 87×10⁶, 88×10⁶, 89×10⁶, 90×10⁶, 91×10⁶, 92×10⁶,93×10⁶, 94×10⁶, 95×10⁶, 96×10⁶, 97×10⁶, 98×10⁶, 99×10⁶, 100×10⁶,101×10⁶, 102×10⁶, 103×10⁶, 104×10⁶, 105×10⁶, 106×10⁶, 107×10⁶, 108×10⁶,109×10⁶, or 110×10⁶) viable CD19 specific CAR-T cells. In someembodiments, the dose is a therapeutically effective amount of viableCD19 specific CAR-T cells. In other embodiments, the dose is aclinically effective amount of viable CD19 specific CAR-T cells. In someembodiments, the viable CD19 specific CAR-T cells include CD19 specificCAR expressing CD4+ T cells and CD19 specific CAR expressing CD8+ Tcells at a ratio of about 1:1. In some embodiments, the CD19 specificCAR of the cells is lisocabtagene maraleucel (BREYANZI©), a structuralequivalent thereof, or a functional equivalent thereof.

In some embodiments, a subject is administered about 50×10⁶ to about110×10⁶ (e.g., 50×10⁶, 51×10⁶, 52×10⁶, 53×10⁶, 54×10⁶, 55×10⁶, 56×10⁶,57×10⁶, 58×10⁶, 59×10⁶, 60×10⁶, 61×10⁶, 62×10⁶, 63×10⁶, 64×10⁶, 65×10⁶,66×10⁶, 67×10⁶, 68×10⁶, 69×10⁶, 70×10⁶, 71×10⁶, 72×10⁶, 73×10⁶, 74×10⁶,75×10⁶, 76×10⁶, 77×10⁶, 78×10⁶, 79×10⁶, 80×10⁶, 81×10⁶, 82×10⁶, 83×10⁶,84×10⁶, 85×10⁶, 86×10⁶, 87×10⁶, 88×10⁶, 89×10⁶, 90×10⁶, 91×10⁶, 92×10⁶,93×10⁶, 94×10⁶, 95×10⁶, 96×10⁶, 97×10⁶, 98×10⁶, 99×10⁶, 100×10⁶,101×10⁶, 102×10⁶, 103×10⁶, 104×10⁶, 105×10⁶, 106×10⁶, 107×10⁶, 108×10⁶,109×10⁶, or 110×10⁶) viable CD19 specific CAR-T cells described herein.In some embodiments, the dose is a therapeutically effective amount ofviable CD19 specific CAR-T cells. In other embodiments, the dose is aclinically effective amount of viable CD19 specific CAR-T cells. In someinstances, 50% of the viable CD19 specific CAR-T cells are CD19 specificCAR expressing CD4+ T cells and 50% of the viable CD19 specific CAR-Tcells are CD19 specific CAR expressing CD8+ T cells. In someembodiments, the CD19 specific CAR of the cells is lisocabtagenemaraleucel (BREYANZI®), a structural equivalent thereof, or a functionalequivalent thereof.

In some embodiments, the CD19 specific CAR-T cells described herein areadministered to a subject at a dose of about 2×10⁶ per kg of bodyweight. In some embodiments, a maximum dose administered is about 2×10⁸viable CD19 specific CAR-T cells. In some embodiments, the dose is atherapeutically effective amount of viable CD19 specific CAR-T cells. Inother embodiments, the dose is a clinically effective amount of viableCD19 specific CAR-T cells. In some embodiments, the CD19 specific CAR ofthe cells is the same CD19 specific CAR as axicabtagene ciloleucel(YESCARTA®), a structural equivalent thereof, or a functional equivalentthereof.

In some embodiments, the CD19 specific CAR-T cells described herein areadministered to a subject at a dose of about 2×10⁶ per kg of bodyweight. In some embodiments, a maximum dose of about 2×10⁸ viable CD19specific CAR-T cells is administered to a patient of about 100 kg ofbody weight and above. In some embodiments, the dose is atherapeutically effective amount of viable CD19 specific CAR-T cells. Inother embodiments, the dose is a clinically effective amount of viableCD19 specific CAR-T cells. In some embodiments, the CD19 specific CAR ofthe cells is the same CD19 specific CAR as brexucabtagene autoleucel(TECARTUS©), a structural equivalent thereof, or a functional equivalentthereof.

In some embodiments, the CD19 specific CAR-T cells described herein areadministered to a subject at a dose of up to about 2×10⁸ viable CD19specific CAR-T cells. In some embodiments, a subject is administeredfrom about 0.2×10⁶ to about 5.0×10⁶ (e.g., about 0.2×10⁶, 0.4×10⁶,0.5×10⁶, 0.6×10⁶, 0.8×10⁶, 0.9×10⁶, 1.0×10⁶, 1.2×10⁶, 1.4×10⁶, 1.5×106,1.6×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.2×10⁶, 2.4×10⁶, 2.5×10⁶, 2.6×10⁶,2.8×10⁶, 2.9×10⁶, 3.0×10⁶, 3.2×10⁶, 3.4×10⁶, 3.5×10⁶, 3.6×10⁶, 3.8×10⁶,3.9×10⁶, 4.0×10⁶, 4.2×10⁶, 4.4×10⁶, 4.5×10⁶, 4.6×10⁶, 4.8×10⁶, 4.9×10⁶,or 5.0×10⁶) viable CD19 specific CAR-T cells per kg of body weight for asubject with a body weight of about 50 kg or less. In some embodiments,a subject is administered from about 0.1×10⁸ to about 2.5×10⁸ (e.g.,about 0.1×106, 0.2×10⁶, 0.4×10⁶, 0.5×10⁶, 0.6×10⁶, 0.8×10⁶, 0.9×10⁶,1.0×10⁶, 1.2×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶,2.2×10⁶, 2.4×10⁶, or 2.5×10⁶) viable CD19 specific CAR-T cells for asubject with a body weight of greater than about 50 kg. In someembodiments, a subject is administered from about 0.6×10⁸ to about6.0×10⁸ (e.g., about 0.6×10′, 0.8×10⁸, 0.9×10⁸, 1.0×10⁸, 1.2×10⁸,1.4×10⁸, 1.5×10⁸, 1.6×10⁸, 1.8×10⁸, 1.9×10⁸, 2.0×10⁸, 2.2×10⁸, 2.4×10⁸,2.5×10⁸, 2.6×10⁸, 2.8×10⁸, 2.9×10⁸, 3.0×10⁸, 3.2×10⁸, 3.4×10⁸, 3.5×10⁸,3.6×10⁸, 3.8×10⁸, 3.9×10⁸, 4.0×10⁸, 4.2×10⁸, 4.4×10⁸, 4.5×10⁸, 4.6×10⁸,4.8×10⁸, 4.9×10⁸, 5.0×10⁸, 5.2×10⁸, 5.4×10⁸, 5.5×10⁸, 5.6×10⁸, 5.8×10⁸,5.9×10⁸, or 6.0×10⁸) viable CD19 specific CAR-T cells. In someembodiments, the dose is a therapeutically effective amount of viableCD19 specific CAR-T cells. In other embodiments, the dose is aclinically effective amount of viable CD19 specific CAR-T cells. In someembodiments, the CD19 specific CAR of the cells is the same CD19specific CAR as tisagenlecleucel (KYMRIAH©), a structural equivalentthereof, or a functional equivalent thereof.

In some embodiments, a single dose of any of the CD19 specific CAR-Tcells described herein includes about 50×10⁶ to about 110×10⁶ (e.g.,50×10⁶, 51×10⁶, 52×10⁶, 53×10⁶, 54×10⁶, 55×10⁶, 56×10⁶, 57×10⁶, 58×10⁶,59×10⁶, 60×10⁶, 61×10⁶, 62×10⁶, 63×10⁶, 64×106, 65×10⁶, 66×10⁶, 67×10⁶,68×10⁶, 69×10⁶, 70×10⁶, 71×10⁶, 72×10⁶, 73×10⁶, 74×106, 75×10⁶, 76×10⁶,77×10⁶, 78×10⁶, 79×10⁶, 80×10⁶, 81×10⁶, 82×10⁶, 83×10⁶, 84×106, 85×10⁶,86×10⁶, 87×10⁶, 88×10⁶, 89×10⁶, 90×10⁶, 91×10⁶, 92×10⁶, 93×10⁶, 94×106,95×10⁶, 96×10⁶, 97×10⁶, 98×10⁶, 99×10⁶, 100×10⁶, 101×10⁶, 102×10⁶,103×10⁶, 104×10⁶, 105×10⁶, 106×10⁶, 107×10⁶, 108×10⁶, 109×10⁶, or110×10⁶) viable CD19 specific CAR-T cells. In some embodiments, the doseis a therapeutically effective amount of viable CD19 specific CAR-Tcells. In other embodiments, the dose is a clinically effective amountof viable CD19 specific CAR-T cells. In some embodiments, the viableCD19 specific CAR-T cells include CD19 specific CAR expressing CD4+ Tcells and CD19 specific CAR expressing CD8+ T cells at a ratio of about1:1. In some embodiments, the CD19 specific CAR is the same CD19specific CAR as lisocabtagene maraleucel (BREYANZI®), a structuralequivalent thereof, or a functional equivalent thereof.

In some embodiments, a single dose of any of the CD19 specific CAR-Tcells described herein includes about 2×10⁸ viable CD19 specific CAR-Tcells. In some embodiments, a single infusion bag of any of the CD19specific CAR-T cells described herein includes about 2×10⁸ viable CD19specific CAR-T cells in a cell suspension of about 68 mL. In someembodiments, the CD19 specific CAR is the same CD19 specific CAR asaxicabtagene ciloleucel (YESCARTA®), a structural equivalent thereof, ora functional equivalent thereof.

In some embodiments, a single dose of any of the CD19 specific CAR-Tcells described herein includes about 2×10⁸ viable CD19 specific CAR-Tcells. In some embodiments, a single infusion bag of any of the CD19specific CAR-T cells described herein includes about 2×10⁸ viable CD19specific CAR-T cells in a cell suspension of about 68 mL. In someembodiments, the CD19 specific CAR is the same CD19 specific CAR asbrexucabtagene autoleucel (TECARTUS©), a structural equivalent thereof,or a functional equivalent thereof.

In some embodiments, a single dose of any of the CD19 specific CAR-Tcells described herein includes about 0.2×10⁶ to about 5.0×10⁶ (e.g.,about 0.2×10⁶, 0.3×10⁶, 0.4×10⁶, 0.5×106, 0.6×10⁶, 0.7×10⁶, 0.8×10⁶,0.9×10⁶, 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶,1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶, 2.2×10⁶, 2.3×10⁶, 2.4×106,2.5×10⁶, 2.6×10⁶, 2.7×10⁶, 2.8×10⁶, 2.9×10⁶, 3.0×10⁶, 3.1×10⁶, 3.2×10⁶,3.3×10⁶, 3.4×10⁶, 3.5×10⁶, 3.6×10⁶, 3.7×10⁶, 3.8×10⁶, 3.9×10⁶, 4.0×10⁶,4.1×10⁶, 4.2×10⁶, 4.3×10⁶, 4.4×10⁶, 4.5×10⁶, 4.6×10⁶, 4.7×10⁶, 4.8×10⁶,4.9×10⁶, or 5.0×10⁶) viable CD19 specific CAR-T cells per kg of bodyweight for a subject with a body weight of 50 kg or less. In someembodiments, a single dose of any of the CD19 specific CAR-T cellsdescribed herein includes about 0.1×10⁸ to about 2.5×10⁸ (e.g., about0.1×10⁶, 0.2×10⁶, 0.3×10⁶, 0.4×10⁶, 0.5×10⁶, 0.6×10⁶, 0.7×10⁶, 0.8×10⁶,0.9×10⁶, 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×106, 1.5×10⁶, 1.6×10⁶,1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶, 2.2×10⁶, 2.3×10⁶, 2.4×10⁶,or 2.5×10⁶) viable CD19 specific CAR-T cells per kg of body weight for asubject with a body weight of more than 50 kg. In some embodiments, asingle dose of any of the CD19 specific CAR-T cells described hereinincludes about 0.6×10⁸ to about 6.0×10⁸ (e.g., about 0.6×10⁸, 0.7×10⁸,0.8×10⁸, 0.9×10⁸, 1.0×10⁸, 1.1×10⁸, 1.2×10⁸, 1.3×10⁸, 1.4×10⁸, 1.5×10⁸,1.6×10⁸, 1.7×10⁸, 1.8×10⁸, 1.9×10⁸, 2.0×10⁸, 2.1×10⁸, 2.2×10⁸, 2.3×10⁸,2.4×10⁸, 2.5×10⁸, 2.6×10⁸, 2.7×10⁸, 2.8×10⁸, 2.9×10⁸, 3.0×10⁸, 3.1×10⁸,3.2×10⁸, 3.3×10⁸, 3.4×10⁸, 3.5×10⁸, 3.6×10⁸, 3.7×10⁸, 3.8×10⁸, 3.9×10⁸,4.0×10⁸, 4.1×10⁸, 4.2×10⁸, 4.3×10⁸, 4.4×10⁸, 4.5×10⁸, 4.6×10⁸, 4.7×10⁸,4.8×10⁸, 4.9×10⁸, 5.0×10⁸, 5.1×10⁸, 5.2×10⁸, 5.3×10⁸, 5.4×10⁸, 5.5×10⁸,5.6×10⁸, 5.7×10⁸, 5.8×10⁸, 5.9×10⁸, or 6.0×10⁸) viable CD19 specificCAR-T cells. In some embodiments, a single infusion bag of any of theCD19 specific CAR-T cells described herein includes about 0.6×10⁸ toabout 6.0×10⁸ (e.g., about 0.6×10⁸, 0.7×10⁸, 0.8×10⁸, 0.9×10⁸, 1.0×10⁸,1.1×10⁸, 1.2×10⁸, 1.3×10⁸, 1.4×10⁸, 1.5×10⁸, 1.6×10⁸, 1.7×10′, 1.8×10⁸,1.9×10⁸, 2.0×10⁸, 2.1×10⁸, 2.2×10⁸, 2.3×10⁸, 2.4×10⁸, 2.5×10⁸, 2.6×10⁸,2.7×10⁸, 2.8×10⁸, 2.9×10⁸, 3.0×10⁸, 3.1×10⁸, 3.2×10⁸, 3.3×10⁸, 3.4×10⁸,3.5×10⁸, 3.6×10′, 3.7×10⁸, 3.8×10⁸, 3.9×10⁸, 4.0×10⁸, 4.1×10⁸, 4.2×10⁸,4.3×10⁸, 4.4×10⁸, 4.5×10⁸, 4.6×10⁸, 4.7×10⁸, 4.8×10⁸, 4.9×10⁸, 5.0×10⁸,5.1×10⁸, 5.2×10⁸, 5.3×10⁸, 5.4×10⁸, 5.5×10′, 5.6×10⁸, 5.7×10⁸, 5.8×10⁸,5.9×10′, or 6.0×10⁸) viable CD19 specific CAR-T cells in a cellsuspension of from about 10 mL to about 50 mL. In some embodiments, thedose is a therapeutically effective amount of viable CD19 specific CAR-Tcells. In other embodiments, the dose is a clinically effective amountof viable CD19 specific CAR-T cells. In some embodiments, the CD19specific CAR of the cells is the same CD19 specific CAR astisagenlecleucel (KYMRIAH®), a structural equivalent thereof, or afunctional equivalent thereof.

Y. Methods for Administering Hypoimmunogenic Cells Including T Cells

As is described in further detail herein, provided herein are methodsfor treating a patient with a condition, disorder, or disorder throughadministration of hypoimmunogenic cells, particularly hypoimmunogenic Tcells. As will be appreciated, for all the multiple embodimentsdescribed herein related to the timing and/or combinations of therapies,the administration of the cells is accomplished by a method or routewhich results in at least partial localization of the introduced cellsat a desired site. The cells can be infused, implanted, or transplanteddirectly to the desired site, or alternatively be administered by anyappropriate route which results in delivery to a desired location in thesubject where at least a portion of the implanted cells or components ofthe cells remain viable.

Provided herein are methods for treating a patient with a condition,disorder, or disorder includes administration of a population ofhypoimmunogenic cells (e.g., primary T cells, T cells differentiatedfrom hypoimmunogenic induced pluripotent stem cells, or other cellsdifferentiated from hypoimmunogenic induced pluripotent stem cellsdescribed herein) to a subject, e.g., a human patient. For instance, apopulation of hypoimmunogenic primary T cells such as, but limited to,CD3+ T cells, CD4+ T cells, CD8+ T cells, naïve T cells, regulatory T(Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells,Th17 cells, T-follicular helper (Tfh) cells, cytotoxic T lymphocytes(CTL), effector T (Teff) cells, central memory T (Tcm) cells, effectormemory T (Tem) cells, effector memory T cells that express CD45RA (TEMRAcells), tissue-resident memory (Trm) cells, virtual memory T cells,innate memory T cells, memory stem cell (Tsc), γδ T cells, and any othersubtype of T cell is administered to a patient to treat a condition,disorder, or disorder. In some embodiments, an immunosuppressive and/orimmunomodulatory agent (such as, but not limited to a lymphodepletionagent) is not administered to the patient before the administration ofthe population of hypoimmunogenic cells. In some embodiments, animmunosuppressive and/or immunomodulatory agent is administered at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more before theadministration of the cells. In some embodiments, an immunosuppressiveand/or immunomodulatory agent is administered at least 1 week, 2 weeks,3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeksor more before the administration of the cells. In numerous embodiments,an immunosuppressive and/or immunomodulatory agent is not administeredto the patient after the administration of the cells, or is administeredat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or moreafter the administration of the cells. In some embodiments, animmunosuppressive and/or immunomodulatory agent is administered at least1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks or more after the administration of the cells. In someembodiments where an immunosuppressive and/or immunomodulatory agent isadministered to the patient before or after the administration of thecells, the administration is at a lower dosage than would be requiredfor cells with MHC I and/or MHC II expression and without exogenousexpression of CD47.

Non-limiting examples of an immunosuppressive and/or immunomodulatoryagent (such as, but not limited to a lymphodepletion agent) includecyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil,corticosteroids such as prednisone, methotrexate, gold salts,sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin,thymosin-α and similar agents. In some embodiments, theimmunosuppressive and/or immunomodulatory agent is selected from a groupof immunosuppressive antibodies consisting of antibodies binding to p75of the IL-2 receptor, antibodies binding to, for instance, MHC, CD2,CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-gamma, TNF-alpha, IL-4, IL-5,IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, or CD58, andantibodies binding to any of their ligands. In some embodiments, such animmunosuppressive and/or immunomodulatory agent may be selected fromsoluble IL-15R, IL-10, B7 molecules (e.g., B7-1, B7-2, variants thereof,and fragments thereof), ICOS, and OX40, an inhibitor of a negative Tcell regulator (such as an antibody against CTLA-4) and similar agents.

In some embodiments, where an immunosuppressive and/or immunomodulatoryagent is administered to the patient before or after the administrationof the cells, the administration is at a lower dosage than would berequired for cells with MHC I and/or MHC II expression, TCR expressionand without exogenous expression of CD47. In some embodiments, where animmunosuppressive and/or immunomodulatory agent is administered to thepatient before or after the first administration of the cells, theadministration is at a lower dosage than would be required for cellswith MHC I and MHC II expression, TCR expression and without exogenousexpression of CD47.

In some embodiments, the cells described are co-administered with atherapeutic agent that that binds to and/or interacts with one or morereceptors selected from the group consisting of CD94, KIR2DL4, PD-1, aninhibitory NK cell receptor, and an activating NK receptor. In someinstances, the therapeutic agent binds to a receptor on the surface ofan NK cell, including one or more subpopulations of NK cells. In someembodiments, the therapeutic agent is selected from the group consistingof an antibody and fragments and variants thereof, an antibody mimetic,a small molecule, a blocking peptide, and a receptor antagonist.

For therapeutic application, cells prepared according to the disclosedmethods can typically be supplied in the form of a pharmaceuticalcomposition comprising an isotonic excipient, and are prepared underconditions that are sufficiently sterile for human administration. Forgeneral principles in medicinal formulation of cell compositions, see“Cell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy,” by Morstyn & Sheridan eds, Cambridge University Press,1996; and “Hematopoietic Stem Cell Therapy,” E. D. Ball, J. Lister & P.Law, Churchill Livingstone, 2000. The cells can be packaged in a deviceor container suitable for distribution or clinical use.

IV. EXAMPLES Example 1: Generation of TRAC, B2M, and CIITA TripleKnockout, CD47 Transgenic CAR-T Cells (tKO/CD47 CAR-T Cells)

Described herein is an exemplary method for producing TRAC, B2M andCIITA triple knockout, CD47 transgenic CAR-T cells (HIP CD19-CAR-Tcells). CD19-specific chimeric antigen receptors (CD19-CAR) wereintroduced in T cells using lentiviral expression technology and theTRAC, B2M and CIITA genes were inactivated using CRISPR/Cas9 technology.

Production of lentiviral vectors: HEK293LX grown in suspension cultureswere transfected using standard chemical transfection complexescontaining viral expression and transfer vectors harboring a control CARor a test CAR, respectively. Cell cultures were harvested and clarifiedpost transfection, followed by centrifugation for concentration.Lentiviral pelleted were resuspended to a final concentrate.

Culturing, transduction, and nucleofection of human pan T cells(including CD3-positive T cells): Pan T cells were thawed and activatedthe same day. After activation, the lentivirus concentrate was mixedwith T cells in standard culture plates. In particular, T cells weremixed with media containing the lentiviral concentrate and thenspinfected. Cells of the same condition were pooled at seeded onnon-treated culture plates. Specifically, T cells were transduced witheither (1) a control construct containing a CD47 transgene alone, (2) acontrol construct containing a CD19-CAR, or (3) a construct containingboth a CD47 transgene and a CD19-CAR (also referred to as “CAR-CD47”).Post transduction, all transduced T cells were collected and pelleted inpreparation for nucleofection of Cas9 ribonucleoproteins (RNPs) togenerate TRAC, B2M, and CIITA triple knockout cells. RNPs for eachtarget locus were complexed separately at a particular sgRNA:Cas9 ratio,followed by mixture of the three RNPs at specific amount per millioncells. The RNP mixture was diluted in a solution, mixed with the cellpellet, and nucleofected under specific conditions. Nucleofected cellswere seeded and cultured, and then cryopreserved post-activation in acryopreservation media. See Table 21 for more information.

TABLE 21 Summary of Test Materials Cell Name Editing Unedited T cellsHLA⁺ TCR⁺ CD47wt CD19-CAR-T cells HLA⁺ TCR⁺ CD47wt CD19- CAR⁺ EGFRe tKOCD19-CAR-T cells HLA⁻ TCR⁻ CD47wt CD19- CAR⁺ EGFRt HIP CD19-CAR-T HLA⁻TCR⁻ CD47⁺⁺ CD19- (tKO/CD47 CD19-CAR-T) CAR⁺ Abbreviations: HLA, humanleukocyte antigen; TCR, T cell receptor; wt, wild-type; −, negative; ++,overexpression; +, positive

Example 2: Creating Hypoimmunogenic CAR-T Cells to Evade ImmuneRecognition for Allogenic Therapies

Off-the-shelf CAR-T cells could offer advantages over autologousstrategies, including ease of manufacturing, quality control andavoidance of malignant contamination and T cell dysfunction. However,the vigorous host-versus-graft immune response against histoincompatibleT cells prevents expansion and persistence of allogeneic CAR-T cells andmitigates the efficacy of this approach.

Described herein are engineered or modified human immune evasive CAR-Tcells based, in part, on the hypoimmune editing platform described inWO2018132783. The inventors have taken advantage of the findings that Tcells lose their immunogenicity when human leukocyte antigen (HLA) classI and II genes are inactivated and CD47 is over-expressed, and that therisk of graft-versus-host disease can be controlled via TCR knockout.Additionally, TCR knockout was performed to control the risk ofgraft-versus-host disease.

This example describes hypoimmunogenic CD19-specific CAR-T cells and theeffect of exogenous CD47 expression on the activity of such cellscompared to control CD19-specific CAR-T cells in in vitro tumor efficacyexperiments. In the experiments, CD19+ tumor cells were used as thetarget cell and CAR-T cells such as the test and control CAR-T cellswere used as the effector cell.

In the study, the test hypoimmunogenic CAR-T cells included (a) genomeediting of the B2M, CIITA and TRAC genes and (b) transgenes containing apolynucleotide encoding a CD19-specific CAR and a polynucleotideencoding CD47. In some instances, the control CAR-T cell included apolynucleotide encoding a CD19-specific CAR. In certain instances, thecontrol CAR-T cell included a T cell expressing the same CAR constructas tisagenlecleucel or a biosimilar/surrogate thereof.

When transplanted into allogeneic humanized mice, hypoimmunogenicHLA-I/II-negative, TCR-negative, CD47-positive, CD19-specific CAR-Tcells evaded immune recognition by T and B cells compared toCD19-specific CAR-T cells generated from the same human donor. Innateimmune cell assays showed that CD47 overexpression protects MHC-I/IIdeficient CAR-T cells from innate immune cell killing in vitro and invivo. CD47 expression levels were analyzed using method for flowcytometric estimation of antibodies per cell to understand thresholdlevels for protection. The strategy described herein could additionallybe used as a safety strategy for the hypoimmunogenic CAR-T cells.

Neither isolated CD47 overexpression nor all three hypoimmune edits orknockouts (B2M/CIITA/TRAC) showed any effect on the cytotoxic potentialof CAR-T cells. Hypoimmunogenic CD19-specific CAR-T cells (e.g.,B2M^(−/−), CIITA^(−/−), TRAC^(−/−) CD19-specific CAR-CD47 T cells)retained their antitumor activity in the CD19+ tumor model in vitro aswell as in NSG mice across a range of tumor cell:CAR-T cell ratios. Itdoes not appear that the introduction of the hypoimmune gene edits(e.g., B2M/CIITA/TRAC gene inactivation) changed their cytokineindependent growth compared to immunogenic CAR-T cells. These findingsshow that hypoimmunogenic CD19-specific CAR-T cells are functionallyimmune evasive in allogeneic recipients with prolonged cytotoxicanti-tumor capacity and suggest they could provide universalimmunotherapeutic options for cancer patients.

In summary, provided in this example and corresponding figures arehypoimmunogenic T cells with CD19-specific CAR activity (e.g., tripleTRAC, B2M, CIITA knockout and both CD19-specific CAR and CD47 proteinoverexpression). Such hypoimmunogenic CAR-T cells are protected frominnate immune cell killing, as demonstrated in in vitro assays. T cellsexogenously expressing a construct containing the CD19-specific CAR andCD47 under the control of a single EF1α promoter (CAR-CD47 construct)showed similar activity as other comparable CD19-specific CAR-T cells,as shown in vitro and in vivo assays. Also, expression of the CAR-CD47construct in T-lymphoblastic cell line (Mo) cells showed cytokineindependent growth. The hypoimmunogenic CAR-T cells described hereinwere not rejected, as shown in vitro and in vivo experiments. Thehypoimmunogenic CAR-T cells also showed a similar tumor killing activityto comparable immunogenic CAR-T cells in both in vitro and in vivoassays. The experimental data is provided in FIGS. 1-30.

Example 3: Absence of Cytokine-Independent Proliferation of TRAC, B2M,and CIITA Triple Knockout CAR-T Cells

On Day −1 the cells were resuspended in culture media supplemented withIL-2. The cells were stained with cell viability dyes to detect live anddead cells using a standard protocol known to those skilled in the art.The stained cells were then plated at a predetermined concentration andincubated at 37° C.; 5% CO2 overnight. On Day 0 the cells were cellswere stained and then counted. The cells were resuspended at apre-selected concentration in culture media without IL-2. The cells weresplit into two aliquots—sample #1 was supplemented with IL-2 and sample#2 was not supplemented with IL-2. The two samples were fed every threedays such that sample #1 was fed with media supplemented with IL-2 andsample #2 was fed with media without IL-2 supplementation. On Day 19both samples were cultured in media supplemented with IL-2. On Day 22the samples were stained with cell viability dyes. The cells werecounted and viability was evaluated using a standard cellometer todetermine the number of live cells and dead cells. FIG. 31 provides aschematic diagram of the experimental approach.

The results show that sample #1 cells (supplemented with IL-2)proliferated and sample #2 cells (not supplemented with IL-2) did not(FIG. 32A and FIG. 32B).

Example 4: tKO/CD47 CAR-T Cell Mediated Cytotoxicity of Nalm6 TumorCells in a Xenograft Mouse Model

NSG mice xenografted with Nalm6-luc leukemia tumor cells were used forevaluating the cytotoxicity of hypoimmunogenic CAR-T cells(HLA-/TCR-/CD47tg CD19-specific CAR-T cells, also referred to astKO/CD47 CAR-T cells or as HIP CD19-CAR-T cells) of the presentdisclosure against the tumor cells. The experimental approach used inthe study is provided in FIG. 33.

Nalm6-luc cells were originally derived from a male acute Blymphoblastic leukemia (B-ALL) patient and have been modified to stablyexpress luciferase and G418 resistance genes. Cells were cultured undersemi-suspension culture conditions and passaged every 2-3 days. For invivo injections, cells were collected while in log-phase of growth andwashed in a standard buffer before administered to the recipient NSGmice.

tKO/CD47 CAR-T (HIP CD19-CAR-T) cells were thawed and cultured usingstandard T cell culture conditions. An aliquot of cells were reservedfor post-thaw flow analysis of viability and CD19-specific CARexpression frequency. Viability and CAR expression was determined usingflow cytometry. Dosing of the CAR-T cells was calculated based on theviability of the cells and frequency of CAR expression.

On day −3 NSG mice were intravenously (iv) injected with Nalm6-luccells. The mice challenged with Nalm6 tumors were distributed into fourtreatment groups. Tumor load was determined over time in miceadministered tKO/CD47 CAR-T (HIP CD19-CAR-T) cells and compared to miceadministered either control CAR-T cells, unedited T cells or saline.

On day 0, a first group of mice was iv injected with tKO/CD47 CAR-T (HIPCD19-CAR-T) cells at three different doses—dose #1 of 1×10⁶ tKO/CD47CAR-T cells (HIP CD19-CAR-T), dose #2 of 3×10⁶ tKO/CD47 CAR-T (HIPCD19-CAR-T) cells and dose #3 of 5×10⁶ tKO/CD47 CAR-T (HIP CD19-CAR-T)cells. A second group of mice was iv injected with control CAR-T cellsat three different doses—dose #1 of 1×10⁶ control CAR-T cells, dose #2of 3×10⁶ control CAR-T cells and dose #3 of 5×10⁶ control CAR-T cells. Athird group of mice was iv injected with 5×10⁶ unedited T cells.Finally, a fourth group of mice was iv injected with saline.

In vivo imaging to detect the tumor cells was performed weekly such ason day 7, 14, 21, 28, and the like over the course of the study. At theendpoint of the study, tissue samples from the mice will be collectedand samples will undergo analysis such as but not limited, cytokineanalysis and viral copy number (VCN).

The images of Nalm6-bearing mice obtained after CAR-T cell therapyshowed delayed tumor growth in tKO/CD47 CAR-T (HIP CD19-CAR-T)cells-treated mice when compared with control CAR-T treated mice,unedited T cell-treated mice and saline-treated mice (FIGS. 34 and 35).

Example 5: Method for Producing Hypoimmunogenic CAR-T Cells forAllogeneic Cell Therapy

This example describes a method of generating hypoimmunogenic CAR-Tcells (B2M^(indel/indel), CIITA^(indel/indel), TRAC^(indel/indel),CD47tg T cells expressing a CD19-specific CAR-T cells, also referred toas tKO/CD47 CD19-CAR-T (HIP CD19-CAR-T) cells) from mononuclear cellsand plasma (leukopak) obtained from healthy female donor who was lessthan 35 years old and had an O+ blood type. The CD4+ and CD8+ T cellsfrom the healthy donor were isolated, separated and activated prior tolentiviral transduction of the CD4+ and CD8+ T cells with lentiviruscarrying a CD47 transgenes and CD19-specific CARs. The transduced cellswere then genetically modified to inactivate the B2M, CIITA and TRACgenes via CRISPR/Cas9, thereby producing tKO/CD47 CD19-CAR-T cells (HIPCD19-CAR-T).

Isolation of CD4+ and CD8+ T cells

To isolate CD4+ and CD8+ T cells from the donor's mononuclearcells/plasma, a serial positive immunomagnetic cell selection strategywas utilized to isolated CD8+ T cells and CD4+ T cells. The CD8+ T cellswere formulated with a cryopreservation media, aliquoted incryopreservation vials, and frozen using a controlled-rate freezer.Separately, the CD4+ T cells were formulated with a cryopreservationmedia, aliquoted in cryopreservation vials, and frozen using acontrolled-rate freezer.

Activation of CD4+ and CD8+ T cells

Frozen aliquots of CD4+ and CD8+ cells were thawed, activated usingCD3/CD28 beads and cultured under conditions for cell growth andexpansion. Briefly, the CD4+ and CD8+ T cells were combined withCD3/CD28 (anti-CD3/anti-CD28) beads at a bead:cell ratio of about 3:1.The cell and bead mixture were seeded at a CD4:CD8 ratio of about 1:1and cultured overnight in a culture media containing IL-2. The processdescribed constituted day 0 of activation.

Lentiviral Vector Production

HEK293-derived cells for lentivirus production were cultured accordingto manufacturer's conditions. At Day 0 (transfection day) the cells weretransfected with a transfection mixture containing lentiviral packagingplasmids and a pre-selected expression vector. At Day 1 aftertransfection, the cells were exposed to an additive such as sodiumbutyrate to improve viral protein production. At Day 1 aftertransfection, viral supernatant was collected, clarified, and thenconcentrated by ultra-high speed centrifugation. The resulting viralpellet was isolated and resuspended in a standard viral formulationbuffer.

Lentiviral transduction of CD4+ and CD8+ T cells

On day 0, frozen aliquots of CD4+ and CD8+ cells were thawed, activatedusing CD3/CD28 beads and cultured under conditions for cell growth andexpansion. Briefly, the CD4+ and CD8+ T cells were combined withCD3/CD28 (anti-CD3/anti-CD28) beads at a bead:cell ratio of about 3:1,seeded at a CD4:CD8 ratio of about 1:1 and cultured overnight in aculture media without cytokines. On the day after activation (day 1post-activation), the activated CD4+ and CD8+ T cells were ready forlentiviral transduction and were divided into three experimental groups.Group 1 cells, considered the untransduced control T cells (“uneditedcontrol”), were allowed to proliferate under standard T cell cultureconditions and were not transduced with a lentiviral vector. Group 2cells, considered the transduced control CD19-CAR-EGFRt T cells(“control CAR-T”), were transduced with a lentiviral vector encoding aCD19-specific CAR and truncated EGFR. Group 3 cells, considered thetransduced hypoimmunogenic CD19-CAR-T cells (“HIP CAR-T”), weretransduced with a lentiviral vector encoding a bicistronic expressioncassette for both CD47 transgenes and a CD19-specific CAR. For Group 2and Group 3 cells, the cells were exposed to their respectiveconcentrated lentiviral particles at a MOI of about 10, as calculatedfrom a functional titer. The cells were transduced using a standardspinfection protocol and were allowed to recover in the incubator forabout 2 days.

After recovery and on day 3 post-activation, the transduced cells (Group2 and 3 cells) and control cells (Group 1 cells) were separatelyharvested and were subjected a CD3/CD28 bead removal method. Theresulting cells from each group were washed and resuspended in a culturemedia containing IL-2.

Gene editing lentiviral transduced CD4+ and CD8+ T cells

Ribonucleoproteins (RNPs) for the TRAC, B2M and CIITA genes wereassembled such that the sgRNAs were precomplexed with recombinant Cas9.Briefly, synthetic modified sgRNAs were mixed with SPyFi Cas9 at a ratiosuch that Cas9 was in excess. Binary complex formation of gRNAstargeting TRAC, CIITA and B2M with Cas9 was carried out according tostandard protocols recognized by those skilled in the art. The RNPs werecombined for the triple knock-out nucleofection at a 1:1.5:2 ratio ofTRAC-RNP:B2M-RNP:CIITA-RNP and nucleofected into the Group 3 cells. Thecells were allowed to recover in the incubator for about 2 days.

Cells of Group 3, as well as those of Groups 1 and 2 were cultured andmaintained using standard protocols known in the art.

Depletion of CD3+ T Cells from HIP CAR-T Cells

On day 8 post-activation, Group 3 “HIP CAR-T” cells were depleted of CD3positive cells using a standard negative selection method known to thoseskilled in the art.

The collected CD3-negative HIP CAR-T cells were resuspended and culturedin culture media supplemented with IL-2. For cryopreservation, the cellswere counted and suspended at a pre-selected concentration in acryopreservation formulation. The cells were stored in liquid nitrogenfor long-term storage.

Characterization of CD3-Depleted HIP CAR-T Cells

On day 10 post-activation cells from experimental Groups 1, 2 and 3 wereprepared for flow cytometry analysis using standard protocols todetermine the cell viability and expression of cell markers includingCD3, CD4, CD8a, HLA-ABC, HLA-DR/DP/DQ, CD19-CAR and CD47. FIG. 38provides a table of flow cytometry results for the HIP CAR-T cells,control CAR-T cells and unedited T cells produced from the methoddescribed.

Results

The data show that the HIP CAR-T cells included a significantly higherpercentage of CD47 positive cells, CD47 positive and CAR positive cells,CD3 negative cells, HLA-ABC negative cells, HLA-DR/DP/DQ negative cells,and triple knockout cells compared to the control CAR-T cells and theunedited T cells. The HIP CAR-T cells contained similar percentages ofCD4+ T cells and CD8+ T cells. The example describes an illustrativemethod for generating hypoimmunogenic CD19-specific CAR-T cells that areuseful for allogeneic CAR-T therapies.

Example 6: Adaptive Immune Evasion by HIP CAR-T Cells in Humanized Mice

Humanized NSG-SGM3 mice were used as recipients for a CDC killing assay.Animals were randomly assigned to experimental groups. 1×10⁶ CD19-CAR-Tcells were injected intravenously into the tail vein, and blood serumwas collected 7 days after injection.

CDC killing assays were performed on the XCelligence MP platform(Agilent Technologies, Santa Clara, Calif.). 96-well E-plates (AgilentTechnologies) were coated with tumor coating solution (AgilentTechnologies) and 4×10⁴ target CD19-CAR-T cells or HIP CD19-CAR-T cellswere plated in 100 μl Optimizer Media (including supplement, ThermoFisher) with human IL-2. After the cell index reached 0.7, 50 μl serumsamples were mixed with 50 μl human complement (Quidel, San Diego,Calif.) and added to the target cells. As killing control, cells weretreated with 2% TritonX100 (data not shown). Data were standardized andanalyzed with the RTCA software (Agilent Technologies).

As shown in FIGS. 39A-B, transplantation of non-hypoimmune editedCD19-CAR-T cells (T cells engineered to express a CAR but lacking anymodifications to B2M, CIITA, or a CD47 transgene) into allogeneichumanized mice resulted in a significant T cell activation, whereashypoimmune CD19-CAR-T cells (T cells engineered to express a CAR and aCD47 transgene, and engineered for reduced expression of B2M and CIITA)evaded immune recognition (p<0.0001).

As shown in FIGS. 39C-D, hypoimmune CD19-CAR-T cells were functionallyimmune evasive in allogeneic humanized mouse recipients, even after CARsensitization. These data suggest that hypoimmune CD19-CAR-T cells canbe used in sensitized patients and for redosing strategies.

Example 7: Generation of Exemplary HIP CD19-CAR-T Cells

HIP CD19-CAR-T cells were manufactured via an ex vivo process includingtwo separate steps for genome modification of purified CD8+ and CD4+ Tcells: a lentiviral transduction step to enable transgene expression ofthe anti-CD19 chimeric antigen receptor (CD19-CAR) and CD47 genes, and agenome editing step in which Cas enzyme is used to target three loci forknockout (TRAC, B2M, and CIITA). The lentiviral vector was pseudotypedwith VSV-G and carried a bicistronic transgene containing both CD19-CARand CD47 genes. The reagents used in the genome editing step comprise anmRNA which encodes the Cas nuclease, or a Cas protein, and three singleguide RNAs (sgRNAs), each of which target one of the loci describedabove. Through (i) CD47 overexpression to block host innate immunity;(ii) reduced expression of MHC class I and class II HLAs to prevent hostadaptive immunity and (iii) reduced expression of T cell receptor (TCR)to prevent graft-versus-host reaction, the HIP CD19-CAR-T cells weredesigned to evade the immune system of the host while mediating thedurable killing of CD19-expressing tumor cells. Previously describedexperiments have demonstrated that these cells are able to hide from theimmune system and avoid immune rejection. See, e.g., PCT/US21/45822,filed Aug. 12, 2021, and WO2021222285, incorporated herein by referencein their entireties.

Both in vitro and in vivo studies using HIP CD19-CAR-T cells havedemonstrated the intended pharmacological activity of targeting andkilling CD19+ tumor cells (NALM-6) that is similar to CD19-CAR-T cells(transduction with a lentiviral vector to express CD19-CAR but nohypoimmune edits). HIP CD19-CAR-T cells lack a graft-versus-hostresponse that is similar to triple knockout (tKO) CD19-CAR-T cells, anduniquely evade innate and adaptive host immune system responses whencompared to standard or tKO CD19-CAR-T cells. See, e.g., PCT/US21/45822,filed Aug. 12, 2021, and WO2021222285, and FIGS. 39 and 41.

Example 8: In Vitro Assessment of the HIP CD19-CAR-T Cell Killing

The objective of this in vitro study was to test the killing activity ofHIP CD19-CAR-T cells described herein.

Cells used in this study included (iHIP CD19-CAR-T cells, (ii)CD19-CAR-T cells, and (iii) unedited T cells from the same donor. Toevaluate killing activity, CD19-CAR-T and HIP CD19-CAR-T cells werethawed, rested overnight, and assessed by flow cytometry for CD19-CARfrequency. CD19-CAR-T and HIP CD19-CAR-T cells were co-cultured for 18hours with NALM-6 cells (target cells labeled with Cell Trace Violet,CTV) at different effector to target ratios (range 3:1 to 1:243, 3-folddilution series). Unedited T cells were added to each E:T ratio tomaintain the same total number of T cells in each condition. Theco-culture of unedited T cells and NALM-6 cells (CTV-labeled) were usedto normalize cell killing.

CD19-CAR-T cells and HIP CD19-CAR-T cells had a similar percentage (75%)of viable cells expressing a CD19-CAR (data not shown). When inco-culture with CD19⁺ NALM-6 tumor target cells, both CD19-CAR-T and HIPCD19-CAR-T cells showed dose-related activity with an effective dose 50%(ED₅₀) of 1:20 effector to target ratio (E:T) (data not shown). Thesefindings indicate that HIP CD19-CAR-T cells have similar expression ofCD19-CAR and demonstrate similar activity to CD19-CAR-T cells.

Example 9: Single Intravenous Dose 108-Day Study of Anti-Tumor Activityof HIP CAR-T Cells in NALM-6-Luc Tumor Bearing Female NSG Mice

The objective of this study was to demonstrate anti-tumor activity ofCas9 gene-editedHIP CD19-CAR-T cells in the CD19⁺ NALM-6-Luciferasetumor NSG mouse model of B-cell malignancy.

Table 22 summarizes this dose-ranging, longitudinal study that includeda tumor rechallenge at Day 60 to evaluate the level of longer-termCD19-CAR-T cell activity.

TABLE 22 Study Groups NALM-6- NALM-6-Luc Luc cells/mouse cells/mouseCD19-CAR⁺ Day 60 Group Day −3 Test Article cells/mouse (Rechallenge) A 1× 10⁶ HIP CD19-CAR-T 5 × 10⁶ 1 × 10⁶ B 1 × 10⁶ HIP CD19-CAR-T 3 × 10⁶ NAC 1 × 10⁶ HIP CD19-CAR-T 1 × 10⁶ NA D 1 × 10⁶ CD19-CAR-T 5 × 10⁶ NA E 1× 10⁶ CD19-CAR-T 3 × 10⁶ NA F 1 × 10⁶ CD19-CAR-T 1 × 10⁶ NA G 1 × 10⁶tKO CD19-CAR-T 5 × 10⁶ 1 × 10⁶ H 1 × 10⁶ Unedited T 5 × 10⁶ NA I 1 × 10⁶Saline NA NA J NA Saline NA 1 × 10⁶ n = 8 for all groups. NA designatesanimals that were not re-challenged, or animals that were not in-life atthe time of the designated bleed. Time of sacrifice varied frommouse-to-mouse.

Female NSG mice (n=8/group) were implanted with 1×10⁶ NALM-6-Luc tumorcells intravenously on Day −3. Viability and CD19-CAR expression on Tcells were evaluated to calculate total T cell numbers (intended numberof CD19-CAR⁺ cell number/% viable, CD19-CAR+ cells). Unedited T cellswere calculated based on frequency of viable cells. On Day 0, CD19-CAR-Tcells were administered to mice. Animals in groups A and G (5×10⁶ HIPCD19-CAR-T cells/mouse and 5×10⁶ tKO CD19-CAR-T cells/mouse,respectively) were re-challenged with an additional intravenousinjection of 1×10⁶ NALM-6-Luc cells on Day 60. A new cohort of naïve NSGmice (group J) was intravenously injected with 1×10⁶ NALM-6-Luc cells toserve as an untreated control.

Immunophenotyping was performed on isolated PBMCs, spleen and bonemarrow T cell populations. Bioluminescence imaging (BLI) was performedto monitor tumor burden on a weekly basis. Histopathology was performedon tissues collected at necropsy and histological changes weresubjectively scored for severity of graft-versus-host response (GvHR),tumor burden and/or other microscopic changes.

Statistical analysis for flow cytometry data was performed with aone-way or two-way analysis of variance (ANOVA) and random coefficientmodels with a random effect for slope were used BLI data and forre-challenged mice.

Results-Tumor Burden. Saline-treated animals were euthanized on Day 21due to moribundity from tumor burden (FIG. 42). Although two unedited Tcell-treated mice had reduced tumor growth, the remaining unedited Tcell-treated mice developed tumors and were sacrificed on Day 35. Two ofeight mice in the 1×10⁶ CD19-CAR-T cell/mouse group had tumor growth andwere sacrificed on Day 35; the remaining six mice in the 1×10⁶CD19-CAR-T group were sacrificed on Day 42 due to GvHR. Animals treatedwith 1×10⁶ HIP CD19-CAR-T cells showed tumor growth starting on Day 21.Mice dosed with 3×10⁶ HIP CD19-CAR-T cells and mice dosed with either5×10⁶ HIP CD19-CAR-T or tKO CD19-CAR-T cells were tumor free until afterNALM-6-Luc rechallenge (Day 60 (FIG. 42)).

Importantly, no HIP CD19-CAR-T or tKO CD19-CAR-T treated animals hadsymptoms of GvHR, whereas every mouse in the 5×10⁶ and 3×10⁶ CD19-CAR-Ttreatment groups developed GvHR symptoms that included significantweight loss, fur loss, and/or hunched posture.

On Day 60, the 5×10⁶ HIP CD19-CAR-T and 5×10⁶ tKO CD19-CAR-T-treatedgroups were re-challenged with 1×10⁶ NALM-6-Luc cells. A new cohort ofnaïve NSG mice injected with 1×10⁶ NALM-6-Luc cells and treated withsaline were added to the study to serve as controls. The 5×10⁶ HIPCD19-CAR-T and tKO CD19-CAR-T animals re-challenged with NALM-6-Luccells displayed similar, significantly improved tumor suppressioncompared to the saline-treated mice over the 48-day period (FIG. 42).The study was terminated on Day 108 when all re-challenged mice hadtumor resurgence.

Results-CD19-CAR-T and CD47 Expression Levels. Frequencies of CD19-CAR⁺cells were evaluated from blood collected at interim bleeds between Days42 and 108 and at time of sacrifice (FIG. 43). Since time of sacrificewas not pre-determined for any group, head-to-head comparisons were notpossible at every timepoint. There was a significant decrease inCD19-CAR⁺ cell frequency in blood of all hypoimmune and tKO CD19-CAR-Ttreated mice, and in 1×10⁶ CD19-CAR-T treated mice, between days 42 and66 (5×10⁶ HIP CD19-CAR-T treated p<0.0001, 3×10⁶ HIP CD19-CAR-T treatedp<0.0001, 1×10⁶ HIP CD19-CAR-T treated p=0.0015; 5×10⁶ tKO CD19-CAR-Ttreated p<0.0001; 1×10⁶ CD19-CAR-T treated p=0.0410). The re-challenged5×10⁶ HIP CD19-CAR-T treated group had a significant increase inCD19-CAR⁺ cells between Days 66 and 108 (p<0.0001), whereas there-challenged 5×10⁶ tKO CD19-CAR-T treated groups did not (p=0.4694).

The CD47MFI was significantly higher on HIP CD19-CAR-T cells than on tKOCD19-CAR-T cells on Day 108 (p<0.0001) (FIG. 43). CD47MFI could not becompared between hypoimmune versus CD19-CAR-T cells due to lack ofsufficient same-day bleeds or sacrifice.

Results. Histopathological analyses were performed on lung, liver, andskin. GvHR characterized by perivascular, peribronchiolar andperiadnexal mononuclear (lymphocytes and macrophages) infiltrates in theliver, lungs, and skin was observed in mice administered CD19-CAR-Tcells or unedited CD19-CAR-T cells. GvHR was not evident in miceadministered HIP CD19-CAR-T or tKO CD19-CAR-T at any dose. Infiltratesof tumor (NALM-6) cells within hepatic sinusoids and/or alveolar septaof lungs were observed in mice administered the lowest (1×10⁶) dose ofHIP CD19-CAR-T cells and in all re-challenged mice. Two miceadministered unedited CD19-CAR-T cells had infiltrates of NALM-6 tumorcells and GvHR concurrently in the liver. Abdominal masses from severalmice treated with 5×10⁶ HIP CD19-CAR-T cells, 1×10⁶ CD19-CAR-T cells, or5×10⁶ tKO CD19-CAR-T cells corresponded to NALM-6 tumor cells that hadreplaced the normal ovarian architecture.

Conclusions. Administration of HIP CD19-CAR-T cells to NSG miceengrafted with CD19⁺ NALM-6-Luc tumor cells resulted in a dose-dependentreduction of systemic tumor burden without evidence of GvHR over a108-day study period. CD19-CAR-T cells at the 3×10⁶ and 5×10⁶ doses alsohad reduced tumor burden, but also developed GvHR as confirmed byhistopathological analysis. The high incidence of GvHR in CD19-CAR-Ttreated animals is likely due to TCR and MHC signaling, which did notoccur in hypoimmune or tKO CD19-CAR-T cells. Furthermore, 5×10⁶ HIPCD19-CAR-T and 5×10⁶ tKO CD19-CAR-T cell/mouse treatments resulted incomparable tumor burden reduction, as well as a significant and similarsuppression when rechallenged with tumor cells compared to salinetreated controls. At study termination, CD19-CAR+ cells were stilldetectable in re-challenged animals, and CD47 expression wassignificantly higher on HIP CD19-CAR-T cells compared to tKO CD19-CAR-Tcells.

Example 10: In Vitro Cytokine-Independent Proliferation of HIPCD19-CAR-T Cell

The objective of this study was to determine whether HIP CD19-CAR-Tcells have the ability to proliferate in the absence of IL2 as anindicator of potential oncogenic transformation.

CD19-CAR-T (control) and HIP CD19-CAR-T cells cultured in the presenceof IL2 showed a similar increased cell count compared to Day 0,suggesting comparable cell proliferation. CD19-CAR-T cells and HIPCD19-CAR-T cells cultured in absence of IL2 were comparable and showedminimal evidence of proliferation, reduced survival (decreased cellcount) and eventually no viable cells were present by day 12 (FIG. 44).

The HIP CD19-CAR-T cells did not demonstrate proliferation in absence ofIL2, suggesting the genomic modifications do not result in potentialoncogenic transformation. Further, HIP CD19-CAR-T cells demonstratedsimilar behavior when compared to CD19-CAR-T cells in the presence orabsence of IL2.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various embodiments from different headings and sections asappropriate according to the spirit and scope of the technologydescribed herein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

1. An engineered cell comprising reduced expression of HLA-A, HLA-B,HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and/or TCR-betarelative to a wild-type cell or a control cell, the engineered cellfurther comprising a set of exogenous polynucleotides comprising a firstexogenous polynucleotide encoding CD47 and a second exogenouspolynucleotide encoding a chimeric antigen receptor (CAR), wherein thefirst and/or second exogenous polynucleotides are inserted into at leastone allele of the cell. 2-5. (canceled)
 6. The engineered cell of claim1, wherein the first exogenous polynucleotide encoding CD47 and thesecond exogenous polynucleotide encoding the CAR are inserted into thesame locus. 7-12. (canceled)
 13. The engineered cell of claim 1, whereinthe CAR is selected from the group consisting of a CD19-specific CAR anda CD22-specific CAR. 14-16. (canceled)
 17. The engineered cell of claim1, wherein the engineered cell comprises reduced expression of B2Mrelative to a wild-type cell or a control cell.
 18. The engineered cellof claim 17, wherein the engineered cell does not express B2M.
 19. Theengineered cell of claim 1, wherein the engineered cell comprisesreduced expression of CIITA relative to a wild-type cell or a controlcell.
 20. The engineered cell of claim 19, wherein the engineered celldoes not express CIITA.
 21. The engineered cell of claim 1, wherein theengineered cell is selected from the group consisting a pluripotent stemcell, an induced pluripotent stem cell, a T cell differentiated from aninduced pluripotent stem cell, a primary T cell, and a cell derived froma primary T cell, and the engineered cell does not express TCR-alphaand/or TCR-beta. 22-25. (canceled)
 26. The engineered cell of claim 1,wherein the engineered cell is a cell derived from a primary T cell. 27.The engineered cell of claim 26, wherein the cell derived from theprimary T cell is derived from a pool of T cells comprising primary Tcells from one or more donor subjects who are different from a recipientsubject. 28-41. (canceled)
 42. An engineered cell comprising reducedexpression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA,TCR-alpha, and/or TCR-beta relative to a wild-type cell or a controlcell.
 43. The engineered cell of claim 42, wherein the engineered cellcomprises reduced expression of CIITA relative to a wild-type cell or acontrol cell.
 44. The engineered cell of claim 42, wherein theengineered cell does not express CIITA.
 45. The engineered cell of claim42, wherein the engineered cell comprises reduced expression B2Mrelative to a wild-type cell or a control cell.
 46. The engineered cellof claim 45, wherein the engineered cell does not express B2M. 47-48.(canceled)
 49. The engineered cell of claim 42, wherein the engineeredcell overexpresses CD47 relative to a wild-type cell or a control cell.50-53. (canceled)
 54. The engineered cell of claim 42, wherein theengineered cell is a cell derived from a primary T cell.
 55. Theengineered cell of claim 54, wherein the cell derived from the primary Tcell is derived from a pool of T cells comprising primary T cells fromone or more donor subjects who are different from a recipient subject.56-61. (canceled)
 62. The engineered cell of claim 42, wherein theengineered cell is selected from the group consisting a pluripotent stemcell, an induced pluripotent stem cell, a T cell differentiated from aninduced pluripotent stem cell, a primary T cell, and a cell derived froma primary T cell, and the engineered cell is a B2M^(indel/indel),CIITA^(indel/indel), TRAC^(indel/indel), and/or TRB^(indel/indel) cell.63-64. (canceled)
 65. The engineered cell of claim 1, wherein the firstand/or second exogenous polynucleotide is inserted into at least oneallele of the cell using viral transduction.
 66. The engineered cell ofclaim 65, wherein the viral transduction is via a lentivirus based viralvector.
 67. The engineered cell of claim 66, wherein the lentivirusbased viral vector is a pseudotyped, self-inactivating lentiviral vectorthat carries the first and/or second exogenous polynucleotides.
 68. Theengineered cell of claim 66, wherein the lentivirus based viral vectoris a self-inactivating lentiviral vector pseudotyped with a vesicularstomatitis VSV-G envelope, and which carries the first and/or secondexogenous polynucleotides.
 69. A pharmaceutical composition comprising apopulation of the engineered cells of claim 1 and a pharmaceuticallyacceptable additive, carrier, diluent or excipient. 70-81. (canceled)82. A pharmaceutical composition comprising a population of theengineered cells of claim 1, a base solution of CryoStor® CSB at aconcentration of about 75% w/w, about 25% w/w PlasmaLyte-A™, about 0.3%w/v human serum albumin (HSA), and about 7.5% v/v dimethylsulfoxide(DMSO).
 83. The pharmaceutical composition of claim 82, wherein thepopulation of the engineered cells is up to about 8.0×10⁸ cells. 84-93.(canceled)
 94. The pharmaceutical composition of claim 82, wherein thepopulation of engineered cells of the pharmaceutical composition orprogeny thereof exhibit at least 40% survival in a subject after 10 daysfollowing administration. 95-97. (canceled)
 98. A dosage regimen fortreating a disease or disorder in a subject comprising administration ofa pharmaceutical composition comprising a population of engineered cellsof claim 1 and a pharmaceutically acceptable additive, carrier, diluentor excipient, wherein the pharmaceutical composition is administered inabout 1-3 doses. 99-118. (canceled)
 119. A dosage regimen for treating adisease or disorder in a subject comprising administering apharmaceutical composition comprising (i) an engineered cell comprisingreduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M,CIITA, TCR-alpha, and/or TCR-beta, the engineered cell furthercomprising a set of exogenous polynucleotides encoding CD47 and achimeric antigen receptor (CAR), wherein the set of exogenouspolynucleotides are inserted into at least one allele of the cell; and(ii) a pharmaceutically acceptable additive, carrier, diluent orexcipient, wherein the pharmaceutical composition comprises up to about6.0×10⁸ cells. 120-121. (canceled)
 122. A dosage regimen for treating adisease or disorder in a subject comprising administering apharmaceutical composition comprising (i) an engineered cell comprisingreduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M,CIITA, TCR-alpha, and/or TCR-beta; and (ii) a pharmaceuticallyacceptable additive, carrier, diluent or excipient, wherein thepharmaceutical composition comprises up to about 6.0×10⁸ cells. 123-124.(canceled)
 125. A method of treating a cancer in a subject comprisingadministration of the engineered cell claim 1 to the subject. 126.(canceled)
 127. A method of preventing T cell exhaustion in a subjectcomprising administration of the engineered cell of claim 1 to thesubject, wherein the CAR is a CD19-specific CAR or a CD22-specific CAR.128-134. (canceled)
 135. A non-activated T cell comprising reducedexpression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA,TCR-alpha, and/or TCR-beta relative to a wild-type T cell, and a firstexogenous polynucleotide encoding a chimeric antigen receptor (CAR).136. The non-activated T cell of claim 135, wherein the non-activated Tcell is a primary T cell. 137-146. (canceled)
 147. The non-activated Tcell of claim 135, wherein the non-activated T cell further comprises asecond exogenous polynucleotide encoding CD47. 148-152. (canceled) 153.The non-activated T cell of claim 135, wherein the second exogenouspolynucleotide encoding CD47 and the first exogenous polynucleotideencoding the CAR are inserted into the same locus. 154-165. (canceled)166. The non-activated T cell of claim 135, wherein the non-activated Tcell does not express B2M.
 167. (canceled)
 168. The non-activated T cellof claim 135, wherein the non-activated T cell does not express CIITA.169-227. (canceled)
 228. An engineered T cell comprising (a) reducedexpression of B2M, CIITA, and/or T cell receptor (TCR)-alpha relative toa control T cell, (b) increased expression of CD47 relative to thecontrol T cell and encoded by a first exogenous polynucleotide, and (c)expression of a CD19-specific chimeric antigen receptor (CAR) encoded bya second exogenous polynucleotide, wherein the CAR comprises an antigenbinding domain comprising the amino acid sequences of SEQ ID NOs: 21-23and SEQ ID NOs: 26-28, and wherein the first and second exogenouspolynucleotides are inserted into a locus of at least one allele of theengineered T cell.